Anti-gpiib/iiia antibodies or uses thereof

ABSTRACT

The present invention provides antibodies and antigen-binding molecules thereof which specifically bind the α and/or β subunits of the non-active form of the GPIIb/IIIIa receptor. The antibodies and antigen-binding molecules can be genetically fused and/or conjugated to heterologous moieties and used, for example, as targeting moieties. The invention also includes methods for screening for these antibodies, as well as methods of making and methods of using chimeric molecules derived from the antibodies.

BACKGROUND OF THE INVENTION

Clotting factors have been administered to patients to improvehemostasis for some time. The advent of recombinant DNA technology hassignificantly improved treatment for patients with clotting disorders,allowing for the development of safe and consistent proteintherapeutics. For example, recombinant activated factor VII has becomewidely used for the treatment of major bleeding, such as that whichoccurs in patients having hemophilia A or B, deficiency of coagulationFactors XI or VII, defective platelet function, thrombocytopenia, or vonWillebrand's disease.

Although such recombinant molecules are effective, there is a need forimproved versions which localize the therapeutic to sites ofcoagulation, have improved pharmacokinetic properties, have improvedmanufacturability, have reduced thrombogenicity, or have enhancedactivity, or more than one of these characteristics.

Treatment of hemophilia by replacement therapy is targeting restorationof clotting activity. There are plasma-derived and recombinant clottingfactor products available to treat bleeding episodes on-demand or toprevent bleeding episodes from occurring by treating prophylactically.Based on the half-life of these products, treatment regimens requirefrequent intravenous administration. Such frequent administration ispainful and inconvenient. Strategies to extend the half-life of clottingfactors include pegylation (Rostin J, et al., Bioconj. Chem. 2000;11:387-96), glycopegylation (Stennicke H R, et al., Thromb. Haemost.2008; 100:920-8), formulation with pegylated liposomes (Spira J, et al.,Blood 2006; 108:3668-3673, Pan J, et al., Blood 2009; 114:2802-2811) andconjugation with albumin (Schulte S., Thromb. Res. 2008; 122 Suppl4:S14-9).

Recombinant FVIIa (rFVIIa; NOVOSEVEN®) is used to treat bleedingepisodes in (i) hemophilia patients with neutralizing antibodies againstFVIII or FIX (inhibitors), (ii) patients with FVII deficiency, or (iii)patients with hemophilia A or B with inhibitors undergoing surgicalprocedures. NOVOSEVEN® displays poor efficacy. Repeated doses of FVIIaat high concentration are often required to control a bleed, due to itslow affinity for activated platelets, short half-life, and poorenzymatic activity in the absence of tissue factor. Accordingly, thereis an unmet medical need for better treatment and prevention option forhemophilia patients with inhibitors in which the activity of the FVIIaprotein is increased.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides antibodies against GPIIb/IIIa that arecapable of targeting the non-active form of the receptor (see FIG. 2E).The GPIIb/IIIa antibodies and antigen-binding molecules derived fromthese GPIIb/IIIa antibodies can be used, for example, to targettherapeutic molecules (e.g., clotting factors) to the platelet. Inaddition to their use as targeting moieties, GPIIb/IIIa antibodies andantigen-binding molecules thereof of the present invention can be usedfor diagnostics, for example, by conjugation to a detectable label.

The present invention also relates to chimeric molecules comprising theGPIIb/IIIa antibodies and antigen-binding molecules thereof disclosedherein as targeting moieties, and one or more heterologous moieties. Forexample, a chimeric molecule can comprise a heterologous moietycomprising a therapeutic molecule (for example, a procoagulant moleculesuch as the FVIIa clotting factor), and optionally a second heterologousmoiety comprising, for example, a PK enhancing moiety (i.e., a moleculewhich can improve various pharmacokinetic properties, e.g., circulationhalf-life).

The present disclosure provides an antibody or antigen-binding moleculethereof which specifically binds to a GPIIb/IIIa epitope, wherein theantibody or antigen-binding molecule thereof specifically bind to thesame GPIIb/IIIa epitope as an antibody selected from 34D10, 12B2, 2A2,35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6, 13C1, 5C4, 23C10,37C7, 28C2, 9D6, or 28F4. The invention also includes an antibody orantigen-binding molecule thereof which specifically binds to aGPIIb/IIIa epitope, wherein the antibody or antigen-binding moleculethereof competitively inhibits GPIIb/IIIa binding by an antibodyselected from 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10,38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4. Alsoprovided is an antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, wherein the antibody orantigen-binding molecule thereof comprises at least one, at least two,at least three, at least four, or at least five complementaritydetermining regions (CDR) or variants thereof of an antibody selectedfrom the 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8,18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4 antibodies. Insome embodiments, the antibody or antigen-binding molecule thereofcomprises six CDRs or variants thereof of an antibody selected from the34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6,13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4 antibodies.

The present disclosure also provides an antibody or antigen-bindingmolecule thereof which specifically binds to a GPIIb/IIIa epitope,comprising (i) a variable heavy chain CDR-1 (VH-CDR1) sequence at leastabout 60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ IDNOS: 25, 31, 37, 43 or 111; (ii) a variable heavy chain CDR-2 (VH-CDR2)sequence at least about 60%, 70%, 80%, 90%, 95%, or 100% identical toany one of SEQ ID NOS:26, 32, 38, 44, or 112; (iii) a variable heavychain CDR-3 (VH-CDR3) sequence at least about 60%, 70%, 80%, 90%, 95%,or 100% identical to any one of SEQ ID NOS: 27, 33, 39, 45, or 113; (iv)a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%, 70%,80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 28, 34, 40,117, or 114; (v) a variable light chain CDR-2 (VL-CDR2) sequence atleast about 60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQID NOS: 29, 35, 41, 118, or 115; and (vi) a variable light chain CDR-3(VL-CDR3) sequence at least about 60, 70, 80, 90, or 95% identical toany one of SEQ ID NOS: 30, 36, 42, 119, or 116.

Also provided is an antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, comprising (i) a VH-CDR1comprising the consensus sequence XIYAMS wherein X₁ represents aminoacid residues Thr (T), Ser (S), or Ala (A); (ii) a VH-CDR2 comprisingthe consensus sequence SIX₂X₃GX₄X₅TYX₆X₇DSVKX₈ wherein X₂ representsamino acid residues Ser (S) or Asn (N), X₃ represents amino acidresidues Ser (S) or Gly (G), X₄ represents amino acid residues Ser (S)or Gly (G), X₅ represents amino acid residues Ser (S), Asn (N), or Thr(T), X₆ represents amino acid residues Tyr (Y) or Phe (F), X₇ representsamino acid residues Leu (L) or Pro (P), and X₈ represents amino acidsGly (G) or Arg (R); (iii) a VH-CDR3 comprising the consensus sequenceGGDYGYAX₉DY, wherein X₉ represents amino acid residues Leu (L) or Met(M); (iv) a VL-CDR1 comprising the sequence RASSSVNYMY (SEQ ID NO: 28);(v) a VL-CDR2 comprising the sequence YTSNLAP (SEQ ID NO: 29); and, (vi)a VL-CDR3 comprising the sequence QQFSSSPWT (SEQ ID NO: 30). In someembodiments, the antibody or antigen-binding molecule thereof comprises(i) a VH-CDR1 sequence selected from SEQ ID NO: 25, 31, 37, 43, or 111;(ii) a VH-CDR2 sequence selected from SEQ ID NOS: 26, 32, 38, 44, or112; (iii) a VH-CDR3 sequence selected from SEQ ID NOS: 27, 33, 39, 45,or 113; (iv) a VL-CDR1 sequence selected from of SEQ ID NOS: 28, 34, 40,117, or 114; (v) a VL-CDR2 sequence selected from SEQ ID NOS: 29, 35,41, 118, or 115; and (vi) a VL-CDR3 sequence selected from SEQ ID NOS:30, 36, 42, 119, or 116.

The instant disclosure also provides an antibody or antigen-bindingmolecule thereof which specifically binds to a GPIIb/IIIa epitope,comprising a VH comprising an amino acid sequence at least about 80%,85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 1, 3, 5, 7,or 97, and a VL comprising an amino acid sequence at least about 80%,85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 2, 4, 6, 99,or 98. In some embodiments, the antibody or antigen-binding moleculethereof comprises a VH comprising the amino acid sequence of SEQ ID NO:1 and a VL comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the antibody or antigen-binding molecule thereofcomprises a VH comprising the amino acid sequence of SEQ ID NO: 3 and aVL comprising the amino acid sequence of SEQ ID NO: 4. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 5 and a VLcomprising the amino acid sequence of SEQ ID NO: 6. In otherembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 7 and a VLcomprising the amino acid sequence of SEQ ID NO: 99. In otherembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 97 and a VLcomprising the amino acid sequence of SEQ ID NO: 98.

In some embodiments, the antibody or antigen-binding molecule thereofcan bind to a GPIIb/IIIa epitope located in the extracellular domain ofthe alpha subunit of GPIIb/IIIa or in the extracellular domain of theGPIIb/IIIa complex. In some embodiments, the antibody or antigen-bindingmolecule thereof does not compete with fibrinogen for binding toGPIIb/IIIa.

The present disclosure also provides an antibody or antigen-bindingmolecule thereof which specifically binds to a GPIIb/IIIa epitopecomprising (i) a variable heavy chain CDR-1 (VH-CDR1) sequence at leastabout 60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ IDNOS: 46, 52, 120, or 126; (ii) a variable heavy chain CDR-2 (VH-CDR2)sequence at least about 60%, 70%, 80%, 90%, 95%, or 100% identical toany one of SEQ ID NOS: 47, 53, 121, or 127; (iii) a variable heavy chainCDR-3 (VH-CDR3) sequence at least about 60%, 70%, 80%, 90%, 95%, or 100%identical to any one of SEQ ID NOS: 48, 54, 122, or 128; (iv) a variablelight chain CDR-1 (VL-CDR1) sequence at least about 60%, 70%, 80%, 90%,95%, or 100% identical to any one of SEQ ID NOS: 49, 55, 123, or 129;(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 50, 56,124, or 130; and (vi) a variable light chain CDR-3 (VL-CDR3) sequence atleast about 60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQID NO: 51, 57, 125, or 131.

Also provided is an antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, comprising (i) a VH-CDR1comprising the sequence NYLIE (SEQ ID NO: 46); (ii) a VH-CDR2 comprisingthe sequence VINPGSGGTNYNEKFKG (SEQ ID NO: 47); (iii) a VH-CDR3comprising the sequence GRYEWYFDV (SEQ ID NO: 48); (iv) a VL-CDR1comprising the consensus sequence RASQDIX₁₀NYLN wherein X₁₀ representsamino acid residues Ser (S) or Thr (T); (v) a VL-CDR2 comprising thesequence YTSRLHS (SEQ ID NO:50); and (vi) a VL-CDR3 comprising thesequence QQGYTLPYT (SEQ ID NO:51). In some embodiments, the antibody orantigen-binding molecule thereof comprises (i) a VH-CDR1 sequenceselected from SEQ ID NO: 46, 52, 120, or 126; (ii) a VH-CDR2 sequenceselected from SEQ ID NO: 47, 53, 121, or 127; (iii) a VH-CDR3 sequenceselected from SEQ ID NO: 48, 54, 122, or 128; (iv) a VL-CDR1 sequenceselected from SEQ ID NO: 49, 55, 123, or 129; (v) a VL-CDR2 sequenceselected from SEQ ID NO: 50, 56, 124, or 130; and, (vi) a VL-CDR3sequence selected from SEQ ID NO: 51, 57, 125, or 131.

Also provided is an antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, comprising a VH comprisingan amino acid sequence at least about 80%, 85%, 90%, 95%, or 100%identical to any one of SEQ ID NOS: 8, 10, 100, or 102, and a VLcomprising an amino acid sequence at least about 80%, 85%, 90%, 95%, or100% identical to any one of SEQ ID NOS: 9, 11, 101, or 103. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 8, and a VLcomprising the amino acid sequence of SEQ ID NO: 9. In otherembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 10 and a VLcomprising the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the antibody or antigen-binding molecule thereofcomprises a VH comprising the amino acid sequence of SEQ ID NO: 100 anda VL comprising the amino acid sequence of SEQ ID NO: 101. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 102 and a VLcomprising the amino acid sequence of SEQ ID NO: 103. In someembodiments, the antibody or antigen-binding molecule thereof binds to aGPIIb/IIIa epitope located in the extracellular domain of the alphasubunit of GPIIb/IIIa. In some embodiments, the antibody orantigen-binding molecule thereof competes with fibrinogen for binding toGPIIb/IIIa.

The present disclosure also provides an antibody or antigen-bindingmolecule thereof which specifically binds to a gpIIb/IIIa epitope,comprising (i) a variable heavy chain CDR-1 (VH-CDR1) sequence at leastabout 60%, 70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 58; (ii)a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%, 70%,80%, 90%, 95%, or 100% identical to SEQ ID NO: 59; (iii) a variableheavy chain CDR-3 (VH-CDR3) sequence at least about 60%, 70%, 80%, 90%,95%, or 100% identical to SEQ ID NO: 60; (iv) a variable light chainCDR-1 (VL-CDR1) sequence at least about 60%, 70%, 80%, 90%, 95%, or 100%identical to SEQ ID NO: 61; (v) a variable light chain CDR-2 (VL-CDR2)sequence at least about 60%, 70%, 80%, 90%, 95%, or 100% identical toSEQ ID NO: 62; and, (vi) a variable light chain CDR-3 (VL-CDR3) sequenceat least about 60%, 70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO:63.

Also provided is an antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, comprising (i) a VH-CDR1sequence comprising SEQ ID NO: 58; (ii) a VH-CDR2 sequence comprisingSEQ ID NO: 59; (iii) a VH-CDR3 sequence comprising SEQ ID NO: 60; (iv) aVL-CDR1 sequence comprising SEQ ID NO: 61; (v) a VL-CDR2 sequencecomprising SEQ ID NO: 62; and (vi) a VL-CDR3 sequence comprising SEQ IDNO: 63. The present disclosure also provides an antibody orantigen-binding molecule thereof which specifically binds to aGPIIb/IIIa epitope comprising a VH comprising an amino acid sequence atleast about 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12 and aVL comprising an amino acid sequence at least about 80%, 85%, 90%, 95%,or 100% identical to SEQ ID NO: 13. In some embodiments, the antibody orantigen-binding molecule thereof can bind to a GPIIb/IIIa epitopelocated in the extracellular domain of the alpha subunit of GPIIb/IIIa.In some embodiments, the antibody or antigen-binding molecule thereofcompetes with fibrinogen for binding to GPIIb/IIIa.

The instant disclosure also provides an antibody or antigen-bindingmolecule thereof which specifically binds to a GPIIb/IIIa epitope,comprising (i) a variable heavy chain CDR-1 (VH-CDR1) sequence at leastabout 60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ IDNOS: 64, 70, or 135; (ii) a variable heavy chain CDR-2 (VH-CDR2)sequence at least about 60%, 70%, 80%, 90%, 95%, or 100% identical toany one of SEQ ID NOS: 65, 71, or 136; (iii) a variable heavy chainCDR-3 (VH-CDR3) sequence at least about 60%, 70%, 80%, 90%, 95%, or 100%identical to any one of SEQ ID NOS: 66, 72, or 137; (iv) a variablelight chain CDR-1 (VL-CDR1) sequence at least about 60%, 70%, 80%, 90%,95%, or 100% identical to any one of SEQ ID NOS: 67, 132, or 138; (v) avariable light chain CDR-2 (VL-CDR2) sequence at least about 60%, 70%,80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 68, 133, or139; and (vi) a variable light chain CDR-3 (VL-CDR3) sequence at leastabout 60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ IDNOS: 69, 134, or 140.

The invention also includes an antibody or antigen-binding moleculethereof which specifically binds to a GPIIb/IIIa epitope, comprising (i)a VH-CDR1 comprising the sequence SYWIE (SEQ ID NO: 64); (ii) a VH-CDR2comprising the consensus sequence EILPGX₁₄GX₁₅TKYNX₁₆KFKG (SEQ ID NO:187), wherein X₁₄ represents amino acid residues Ser (S) or Thr (T), X₁₅represents amino acid residues Ile (I) or Tyr (Y), and X₁₆ representsamino acid residues Asp (D) or Glu (E); (iii) a VH-CDR3 comprising thesequence LISYYYAMDY (SEQ ID NO: 66); (iv) a VL-CDR1 comprising thesequence RASQDISNYLN (SEQ ID NO: 67); (v) a VL-CDR2 comprising thesequence YTSRLHS (SEQ ID NO: 68); and, (vi) a VL-CDR3 comprising thesequence QQGNTLPPT (SEQ ID NO: 69).

Also provided is an antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, comprising (i) a VH-CDR1sequence selected from SEQ ID NO: 64, 70, or 135; (ii) a VH-CDR2sequence selected from SEQ ID NO: 65, 71, or 136; (iii) a VH-CDR3sequence selected from SEQ ID NO: 66, 72, or 137; (iv) a VL-CDR1sequence selected from SEQ ID NO: 67, 132, or 138; (v) a VL-CDR2sequence selected from SEQ ID NO: 68, 133, or 139; and, (vi) a VL-CDR3sequence selected from SEQ ID NO: 69, 134, or 140.

Also provided herein is an antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope, comprising a VHcomprising an amino acid sequence at least about 80%, 85%, 90%, 95%, or100% identical to any one of SEQ ID NOS: 14, 16, or 105 and a VLcomprising an amino acid sequence at least about 80%, 85%, 90%, 95%, or100% identical to any one of SEQ ID NOs: 15, 104, or 106. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 14 and a VLcomprising the amino acid sequence of SEQ ID NO: 15. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 16 and a VLcomprising the amino acid sequence of SEQ ID NO: 104. In otherembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 105 and a VLcomprising the amino acid sequence of SEQ ID NO: 106. In someembodiments, the antibody or antigen-binding molecule thereof binds to aGPIIb/IIIa epitope located in the extracellular domain of the betasubunit of GPIIb/IIIa. In some embodiments, the antibody orantigen-binding molecule thereof does not compete with fibrinogen forbinding to GPIIb/IIIa.

The present disclosure also provides an antibody or antigen-bindingmolecule thereof which specifically binds to a GPIIb/IIIa epitope,comprising (i) a variable heavy chain CDR-1 (VH-CDR1) sequence at leastabout 60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ IDNOS: 73, 76, 79, 85, or 147; (ii) a variable heavy chain CDR-2 (VH-CDR2)sequence at least about 60%, 70%, 80%, 90%, 95%, or 100% identical toany one of SEQ ID NOS: 74, 77, 80, 86, or 148; (iii) a variable heavychain CDR-3 (VH-CDR3) sequence at least about 60%, 70%, 80%, 90%, 95%,or 100% identical to any one of SEQ ID NOS: 75, 78, 81, 87, or 149; (iv)a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%, 70%,80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 141, 144, 82,88, or 150; (v) a variable light chain CDR-2 (VL-CDR2) sequence at leastabout 60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ IDNOS: 142, 145, 83, 89, or 151; and, (vi) a variable light chain CDR-3(VL-CDR3) sequence at least about 60%, 70%, 80%, 90%, 95%, or 100%identical to any one of SEQ ID NO: 143, 146, 84, 90, or 152. Alsoprovided is an antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, comprising (i) a VH-CDR1comprising the consensus sequence TSGX₁₁GVG, wherein X₁₁ representsamino acid residues Met (M) or Leu (L); (ii) a VH-CDR2 comprising theconsensus sequence HIWWDDDKRYNPX₁₂LKS, wherein X₁₂ represents amino acidresidues Ala (A) or Thr (T); (iii) a VH-CDR3 comprising the consensussequence SHYX₁₃GTFYFDX₁₄, wherein X₁₃ represents amino acid residues Tyr(Y) or Asn (N), and X₁₄ represents amino acid residues Tyr (Y) or Phe(F); (iv) a VL-CDR1 comprising the sequence RASKSISKYLA (SEQ ID NO: 82);(v) a VL-CDR2 comprising the sequence SGSTLQS (SEQ ID NO: 83); and, (vi)a VL-CDR3 comprising the sequence QQHIEYPWT (SEQ ID NO: 84).

In some embodiments, the antibody or antigen-binding molecule thereofcomprises (i) a VH-CDR1 sequence selected from SEQ ID NOS: 73, 76, 79,85, or 147; (ii) a VH-CDR2 sequence selected from SEQ ID NOS: 74, 77,80, 86, or 148; (iii) a VH-CDR3 sequence selected from SEQ ID NOS: 75,78, 81, 87, or 149; (iv) a VL-CDR1 sequence selected from SEQ ID NOS:141, 144, 82, 88, or 150; (v) a VL-CDR2 sequence selected from SEQ IDNOS: 142, 145, 83, 89, or 151; and (vi) a VL-CDR3 sequence selected fromSEQ ID NOS: 143, 146, 84, 90, or 152. Also provided is an antibody orantigen-binding molecule thereof which specifically binds to aGPIIb/IIIa epitope, comprising a VH comprising an amino acid sequence atleast 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS:17, 18, 19, 21, or 109 and a VL comprising an amino acid sequence atleast 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS:107, 108, 20, 22, or 110.

In some embodiments, the antibody or antigen-binding molecule thereofcomprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and aVL comprising the amino acid sequence of SEQ ID NO: 107. In otherembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 18 and a VLcomprising the amino acid sequence of SEQ ID NO: 108. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 109 and a VLcomprising the amino acid sequence of SEQ ID NO: 110.

In some embodiments, the antibody or antigen-binding molecule thereofcomprises a VH comprising the amino acid sequence of SEQ ID NO: 19 and aVL comprising the amino acid sequence of SEQ ID NO: 20. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH comprising the amino acid sequence of SEQ ID NO: 21 and a VLcomprising the amino acid sequence of SEQ ID NO: 22. In someembodiments, the antibody or antigen-binding molecule thereof binds to aGPIIb/IIIa epitope located in the extracellular domain of the betasubunit of GPIIb/IIIa. In some embodiments, the antibody orantigen-binding molecule thereof competes with fibrinogen for binding toGPIIb/IIIa.

Also provided in the present disclosure is an antibody orantigen-binding molecule thereof which specifically binds to aGPIIb/IIIa epitope, comprising (i) a variable heavy chain CDR-1(VH-CDR1) sequence at least about 60%, 70%, 80%, 90%, 95%, or 100%identical to SEQ ID NO: 91; (ii) a variable heavy chain CDR-2 (VH-CDR2)sequence at least about 60%, 70%, 80%, 90%, 95%, or 100% identical toSEQ ID NO: 92; (iii) a variable heavy chain CDR-3 (VH-CDR3) sequence atleast about 60%, 70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 93;(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 94; (v) a variablelight chain CDR-2 (VL-CDR2) sequence at least about 60%, 70%, 80%, 90%,95%, or 100% identical to SEQ ID NO: 95; and (vi) a variable light chainCDR-3 (VL-CDR3) sequence at least about 60%, 70%, 80%, 90%, 95%, or 100%identical to SEQ ID NO: 96.

The present disclosure also provides an antibody or antigen-bindingmolecule thereof which specifically binds to a GPIIb/IIIa epitope,comprising (i) a VH-CDR1 sequence comprising SEQ ID NO: 91; (ii) aVH-CDR2 sequence comprising SEQ ID NO: 92; (iii) a VH-CDR3 sequencecomprising SEQ ID NO: 93; (iv) a VL-CDR1 sequence comprising SEQ ID NO:94; (v) a VL-CDR2 sequence comprising SEQ ID NOS: 95; and (vi) a VL-CDR3sequence comprising SEQ ID NOS: 96.

Also provided is an antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, comprising a VH comprisingan amino acid sequence at least about 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 23 and a VL comprising an amino acid sequence atleast about 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 24. Insome embodiments, the antibody or antigen-binding molecule thereof bindsto a GPIIb/IIIa epitope located in the extracellular domain of the betasubunit of GPIIb/IIIa. In some embodiments, the antibody orantigen-binding molecule thereof competes with fibrinogen for binding toGPIIb/IIIa.

In some embodiments, the antibody or antigen-binding molecule thereofdisclosed herein comprises or consists of (a) a single chain Fv(“scFv”); (b) a diabody; (c) a minibody; (d) a polypeptide chain of anantibody; (e) F(ab′)₂; or (f) F(ab).

The present disclosure also provides a chimeric molecule comprising (i)a GPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein; (ii) a heterologous moiety; and, (iii) an optional linker. Insome embodiments, the chimeric molecule has the formula (i) Tm-(L)-H; or(ii) H-(L)-Tm, wherein, H is a heterologous moiety; L is an optionallinker; and, Tm is an anti-GPIIb/IIIa antibody or antigen-bindingmolecule thereof disclosed herein. In some embodiments, the heterologousmoiety comprises a clotting factor. In some embodiments, the clottingfactor is independently selected from the group consisting of FVII, FIX,FX, and any combinations thereof. In some embodiments, the clottingfactor is FVII zymogen, activatable FVII, activated FVII (FVIIa), FIXzymogen, activatable FIX, activated FIX (FIXa), FX zymogen, activatableFX, or activated FX (FXa). In some embodiments, the chimeric moleculefurther comprises a second heterologous moiety.

In some embodiments, the chimeric molecule has a formula selected from(i) H1-(L1)-Tm-(L2)-H2; (ii) H2-(L2)-Tm-(L1)-H1; (iii)H1-(L1)-H2-(L2)-Tm; (iv) H2-(L2)-H1-(L1)-Tm; (v) Tm-(L1)-H1-(L2)-H2; or(vi) Tm-(L2)-H2-(L1)-H1; wherein, Tm is an anti-GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein; H1 is a firstheterologous moiety, H2 is a second heterologous moiety, L1 is a firstoptional linker, L2 is a second optional linker. In some embodiments,the first heterologous moiety and the second heterologous moiety are thesame or different. In some embodiments, the second heterologous moietycomprises a half-life extending moiety.

In some embodiments, L1 and L2 are the same or different. In someembodiments, the first heterologous moiety comprises a clotting factorand the second heterologous moiety comprises a half-life extendingmoiety. In some embodiments, the second heterologous moiety comprises alow-complexity polypeptide. In some embodiments, the second heterologousmoiety comprises albumin, albumin binding polypeptide or fatty acid, Fc,transferrin, PAS, the C-terminal peptide (CTP) of the β subunit of humanchorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch(HES), albumin-binding small molecules, vWF, a clearance receptor orfragment thereof which blocks binding of the chimeric molecule to aclearance receptor or any combinations thereof. In some embodiments, theclotting factor comprises a single polypeptide chain or two polypeptidechains.

Also provided herein is a chimeric molecule comprising a firstpolypeptide chain and a second polypeptide chain, which are associatedwith each other, (1) wherein the first polypeptide chain comprises alight chain of a clotting factor and a heterologous moiety, and thesecond polypeptide chain comprises a heavy chain of the clotting factorand a GPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein; (2) wherein the first polypeptide chain comprises a light chainof a clotting factor and a GPIIb/IIIa antibody or antigen-bindingmolecule thereof disclosed herein, and the second polypeptide chaincomprises a heavy chain of the clotting factor and a heterologousmoiety; (3) wherein the first polypeptide chain comprises a light chainof a clotting factor and the second polypeptide chain comprises a heavychain of the clotting factor, a GPIIb/IIIa antibody or antigen-bindingmolecule thereof disclosed herein, and a heterologous moiety; (4)wherein the first polypeptide chain comprises a light chain of aclotting factor and the second polypeptide chain comprises a heavy chainof the clotting factor, a heterologous moiety, and a GPIIb/IIIa antibodyor antigen-binding molecule thereof disclosed herein; (5) wherein thefirst polypeptide chain comprises a light chain of a clotting factor, aheterologous moiety, and a GPIIb/IIIa antibody or antigen-bindingmolecule thereof disclosed herein, and the second polypeptide chaincomprises a heavy chain of the clotting factor; or (6) wherein the firstpolypeptide chain comprises a light chain of a clotting factor, aGPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein, and a heterologous moiety and the second polypeptide chaincomprises a heavy chain of the clotting factor.

In some embodiments, the chimeric molecule comprises a first polypeptidechain and a second polypeptide chain, which are associated with eachother: (1) wherein the first polypeptide chain comprises CF_(L)-H orH-CF_(L) and the second polypeptide chain comprises CF_(H)-Tm orTm-CF_(H); (2) wherein the first polypeptide chain comprises CF_(L)-Tmor Tm-CF_(L) and the second polypeptide chain comprises CF_(H)-H orH-CF_(H); (3) wherein the first polypeptide chain comprises CF_(L) andthe second polypeptide chain comprises CF_(H)-Tm-H or H-Tm-CF_(H); (4)wherein the first polypeptide chain comprises CF_(L) and the secondpolypeptide chain comprises CF_(H)-H-Tm or Tm-H-CF_(H); (5) wherein thefirst polypeptide chain comprises CF_(L)-H-Tm or Tm-H-CF_(L) and thesecond polypeptide chain comprises CF_(H); or (6) wherein the firstpolypeptide chain comprises CF_(L)-Tm-H or H-Tm-CF_(L) and the secondpolypeptide chain comprises CF_(H); wherein CF_(L) is a light chain of aclotting factor; CF_(H) is a heavy chain of the clotting factor; Tm isan anti-GPIIb/IIIa antibody or antigen-binding molecule thereofdisclosed herein; or H is a heterologous moiety. In some embodiments,the clotting factor is independently selected from FVII, FIX, and FX.

In other embodiments, the chimeric molecule comprises one or more of thelinkers, wherein one or more of the linkers comprise a peptide linker.In some embodiments, the peptide linker comprises at least two, at leastthree, at least four, at least five, at least 10, at least 20, at least30, at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, or at least 100 amino acids. In some embodiments, the peptidelinker comprises at least 200, at least 300, at least 400, at least 500,at least 600, at least 700, at least 800, at least 900, at least 1000,at least 1100, at least 1200, at least 1300, at least 1400, at least1500, at least 1600, at least 1700, at least 1800, at least 1900, or atleast 2000 amino acids. In some embodiments, the peptide linkercomprises a peptide having the formula [(Gly)_(x)-Ser_(y)]_(z) where xis from 1 to 4, y is 0 or 1, and z is from 1 to 50. In otherembodiments, one or more of the linkers comprise a non-peptide linker.

In some embodiments, the heterologous moiety comprises a half-lifeextending moiety comprising a low-complexity polypeptide. In someembodiments, the chimeric molecule comprises a half-life extendingmoiety comprising albumin, albumin binding polypeptide or fatty acid,Fc, transferrin, PAS, the C-terminal peptide (CTP) of the 1 subunit ofhuman chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethylstarch (HES), albumin-binding small molecules, vWF, a clearance receptoror fragment thereof which blocks binding of the chimeric molecule to aclearance receptor, or any combinations thereof. In some embodiments,the half-life extending moiety comprises an Fc region. In otherembodiments, the half-life extending moiety comprises two Fc regionsfused by a linker. In some embodiments, the chimeric molecule comprisesa clotting factor comprising FVII zymogen, activatable FVII, activatedFVII, FIX zymogen, activatable FIX, activated FIX, FX zymogen,activatable FX, or activated FX.

The instant disclosure also provides a chimeric molecule comprising afirst polypeptide chain and a second polypeptide chain, which areassociated with each other, (1) wherein the first polypeptide chaincomprises a light chain of a clotting factor and a targeting moiety,which binds to a platelet, and the second polypeptide chain comprises aheavy chain of the clotting factor and a heterologous moiety; (2)wherein the first polypeptide chain comprises a light chain of aclotting factor and a heterologous moiety and the second polypeptidechain comprises a heavy chain of the clotting factor and a targetingmoiety, which binds to a platelet; (3) wherein the first polypeptidechain comprises a light chain of a clotting factor, a heterologousmoiety, and a targeting moiety, which binds to a platelet, and thesecond polypeptide comprises a heavy chain of the clotting factor; or(4) wherein the first polypeptide chain comprises a light chain of aclotting factor, a targeting moiety, which binds to a platelet, and aheterologous moiety and the second polypeptide chain comprises a heavychain of the clotting factor, and wherein the clotting factor is FVII,FIX, or FX.

In some embodiments, the chimeric molecule comprises a first polypeptidechain and a second polypeptide chain, which are associated with eachother, (1) wherein the first polypeptide chain comprises CF_(L)-H orH-CF_(L) and the second polypeptide chain comprises CF_(H)-Tm orTm-CF_(H); (2) wherein the first polypeptide chain comprises CF_(L)-Tmor Tm-CF_(L) and the second polypeptide chain comprises CF_(H)-H orH-CF_(H); (3) wherein the first polypeptide chain comprises CF_(L)-H-Tmor Tm-H-CF_(L) and the second polypeptide chain comprises CF_(H); or (4)wherein the first polypeptide chain comprises CF_(L)-Tm-H or H-Tm-CF_(L)and the second polypeptide chain comprises CF_(H); wherein H is aheterologous moiety, CF_(H) is a heavy chain of a clotting factor,CF_(L) is a light chain of the clotting factor, Tm is a targeting moietywhich binds to a platelet, and, L is an optional linker. In someembodiments, the chimeric molecule comprises a formula selected from (1)Tm-CF_(H):CF_(L)-H; (2) H-CF_(H):CF_(L)-Tm; (3) Tm-H-CF_(L):CF_(H); or(4) H-Tm-CF_(L):CF_(H); wherein, H is a heterologous moiety; CF_(H) is aheavy chain of a clotting factor; CF_(L) is a light chain of theclotting factor; Tm is a targeting moiety, which binds to a platelet; Lis an optional linker; and: represents a covalent or non-covalent bondbetween CF_(H) and CF_(L).

In some embodiments, the association between the first polypeptide chainand the second polypeptide chain in the chimeric molecule is a covalentbond or a non-covalent bond. In other embodiment, the associationbetween the first polypeptide chain and the second polypeptide chain inthe chimeric molecule is a covalent bond between the heavy chain and thelight chain of the clotting factor. In some embodiments, the covalentbond is a disulfide bond.

The present disclosure also provides a chimeric molecule comprising asingle polypeptide chain, which comprises, from N terminus to Cterminus, (a) a light chain of a clotting factor, a heterologous moiety,a protease cleavage site, a heavy chain of the clotting factor, and atargeting moiety which binds to a platelet or (b) a light chain of aclotting factor, a targeting moiety which binds to a platelet, aprotease cleavage site, a heavy chain of the clotting factor, and aheterologous moiety, wherein the clotting factor is FVII, FIX, or FX. Insome embodiments, the protease cleavage site is an intracellularprocessing site. In some embodiments, the intracellular processing siteis processed by a proprotein convertase. In some embodiments, theproprotein convertase is selected from PC5, PACE, PC7, and anycombinations thereof.

In some embodiments, the targeting moiety in the chimeric molecule isselected from: an antibody or antigen binding molecule thereof, areceptor binding portion of a receptor, and a peptide. In someembodiments, the targeting moiety selectively binds to a restingplatelet or an activated platelet. In other embodiments, the targetingmoiety selectively binds to a target selected from the group consistingof: GPIba, GPVI, GPIX, a nonactive form of glycoprotein IIb/IIIa(“GPIIb/IIIa”), an active form of GPIIb/IIIa, P selectin, GMP-33,LAMP-1, LAMP-2, CD40L, LOX-1, and any combinations thereof. In certainembodiments, the targeting moiety is an anti-GPIIb/IIIa antibody orantigen-binding molecule thereof. In specific embodiments, theanti-GPIIb/IIIa antibody or antigen-binding molecule thereof is aGPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein. In some embodiments, a heterologous moiety in the chimericmolecule is a half-life extending moiety. In some embodiments, thehalf-life extending moiety is a low-complexity polypeptide. In someembodiments, the half-life extending moiety is selected from albumin,albumin binding polypeptide or fatty acid, Fc, transferrin, PAS, theC-terminal peptide (CTP) of the β subunit of human chorionicgonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES),albumin-binding small molecules, vWF, a clearance receptor or fragmentthereof which blocks binding of the chimeric molecule to a clearancereceptor, and any combinations thereof.

Also provided is a nucleic acid molecule or a set of nucleic acidmolecules encoding an anti-GPIIb/IIIa antibody or antigen-bindingmolecule thereof disclosed herein, or any of the chimeric moleculesdisclosed herein, or a complement thereof. Also provided are a vector ora set of vectors comprising such nucleic acid molecule or the set of thenucleic acid molecules or a complement thereof. Also provided is a hostcell comprising the vector.

The instant disclosure also provides a method for producing ananti-GPIIb/IIIa antibody or antigen-binding molecule thereof or chimericmolecule disclosed herein, such method comprising culturing the hostcell disclosed herein and recovering the antibody, antigen-bindingmolecule thereof, or the chimeric molecule from the culture medium. Alsoprovided is a pharmaceutical composition comprising (i) ananti-GPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein, or (ii) a chimeric molecule disclosed herein, or (iii) a nucleicacid molecule or the set of nucleic acid molecules disclosed herein, or(iv) a vector or set of vectors disclosed herein, and a pharmaceuticallyacceptable carrier. Also provided is method of reducing a frequency ordegree of a bleeding episode in a subject in need thereof comprisingadministering (i) a chimeric molecule disclosed herein, (ii) a nucleicacid molecule or set of nucleic acid molecules disclosed herein, (iii) avector or set of vectors disclosed herein, or (iii) a pharmaceuticalcomposition disclosed herein.

Also provided is a method of reducing or preventing an occurrence of ableeding episode in a subject in need thereof comprising administering(i) a chimeric molecule disclosed herein, (ii) a nucleic acid moleculeor set of nucleic acid molecules disclosed herein, (iii) a vector or theset of vectors disclosed herein, or (iv) a pharmaceutical compositiondisclosed herein. In some embodiments, the subject has developed or hasa tendency to develop an inhibitor against Factor VIII (“FVIII”), FactorIX (“FIX”), or both. In some embodiments, the inhibitor against FVIII orFIX is a neutralizing antibody against FVIII, FIX, or both. In someembodiments, the bleeding episode is caused by a blood coagulationdisorder. In some embodiments, the blood coagulation disorder ishemophilia A or hemophilia B. In some embodiments, the bleeding episodeis derived from hemarthrosis, muscle bleed, oral bleed, hemorrhage,hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis,gastrointestinal bleeding, intracranial hemorrhage, intra-abdominalhemorrhage, intrathoracic hemorrhage, bone fracture, central nervoussystem bleeding, bleeding in the retropharyngeal space, bleeding in theretroperitoneal space, bleeding in the illiopsoas sheath, or anycombinations thereof. In certain embodiments, the subject is a humansubject.

The instant disclosure also provides the chimeric molecule disclosedherein, the nucleic acid molecule or the set of nucleic acid moleculesdisclosed herein, the vector or the set of vectors disclosed herein, orthe pharmaceutical composition disclosed herein for use in reducing afrequency or degree of a bleeding episode or reducing or preventing anoccurrence of a bleeding episode in a subject in need thereof. Alsoprovided is the use of the chimeric molecule disclosed herein, thenucleic acid molecule or the set of nucleic acid molecules disclosedherein, the vector or the set of vectors disclosed herein, or thepharmaceutical composition disclosed herein for the manufacture of amedicament for reducing a frequency or degree of a bleeding episode orreducing or preventing an occurrence of a bleeding episode in a subjectin need thereof.

Also provided is a method to target a therapeutic or prophylactic agentto the surface of platelets comprising fusing the agent to one of theGPIIb/IIIa antibodies or antigen-binding molecules thereof disclosedherein. Also provided is a method to increase the activity of atherapeutic or prophylactic agent comprising fusing the agent to aGPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein. In some embodiments, the therapeutic or prophylactic agent is aclotting factor.

The present disclosure also provides a method to improve thepharmacokinetic properties of a clotting factor comprising fusing theclotting factor to the GPIIb/IIIa antibody or antigen-binding moleculethereof disclosed herein. In some embodiments, the method furthercomprises fusing or conjugating the clotting factor and/or theGPIIb/IIIa antibody or antigen-binding molecule thereof disclosed hereinto a half-life extending moiety.

The present disclosure also provides a method of measuring the level ofplatelets in plasma of a subject in need thereof comprising contactingthe GPIIb/IIIa antibody or antigen binding molecule thereof disclosedherein with the plasma from the subject and measuring the level ofplatelets in plasma.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 is a diagram summarizing the screening process used to identifythe antibodies of the present disclosure.

FIG. 2A shows a diagram of an anti-GPIIb/IIIa antibody. FIGS. 2B, 2C,and 2D are schematic representations of the possible effects of thebinding of antibodies to non-active GPIIb/IIIa. Antibodies can be inert(no activation) (FIG. 2B), activating (FIG. 2C), or capable of indirectactivation via simultaneous interaction with GPIIb/IIIa and the FcγRIIAreceptor (FIG. 2D). Also shown is a schematic representation of thenonactive (bent) and active (extended) conformations of GPIIb/IIIa (FIG.2E). The cross over FIG. 2C indicates that platelet activatingantibodies are not selected as targeting moieties for clotting factors.

FIG. 3A shows the markers detected in the X- and y-axes in the flowcytometry profiles shown in FIGS. 3B, 3C, 3D, and 3E. FIG. 3B shows flowcytometry profiles of platelets exposed to culture medium only or withan agonist in the presence or absence of an FcγRIIA inhibitory antibody.FIG. 3C shows flow cytometry profiles of platelets incubated withhybridoma clone P24H2 supernatant in the presence or absence of anFcγRIIA inhibitory antibody. FIG. 3D shows flow cytometry profiles ofplatelets incubated with hybridoma clone 35E2 supernatant in thepresence or absence of an FcγRIIA inhibitory antibody. FIG. 3E showsflow cytometry profiles of platelets incubated with supernatants fromhybridoma clones P24A4, P31G2, and P35E9, respectively, in the presenceor absence of an FcγRIIA inhibitory antibody.

FIGS. 4A and 4B show identity matrices corresponding to heavy chainvariable domain (VH) sequences (FIG. 4A) and light chain variable domain(VL) sequences (FIG. 4B) of antibodies against GPIIb/IIIa in which theshaded cells indicate which antibodies share identical VL or VHsequences.

FIGS. 5A and 5B show ClustalX multiple sequence alignments of the VHsequences of monoclonal antibodies 1H6, 38A8, 12B2, 38F6, 2A2, 36A8,4B11, 34D10, 28F4, 23C10, 28C2, 5C4, 9D6, and 18F7 (FIG. 5A) and of theVL sequences of monoclonal antibodies 28C2, 9D6, 1H6, 38A8, 12B2, 18F7,28F4, 34D10, 36A8, and 2A2 (FIG. 5B). Degree of amino acid conservationis indicating above the alignment (“*”=identical; “:”=stronglyconserved; “.”=poorly conserved), as well as the bars below thealignment. The ClustalX highlighting scheme, which is dependent on aminoacid physicochemical and/or structural properties and conservation, wasused to highlight the sequences.

FIG. 6 shows a ClustalX multiple sequence alignment of the VH sequencesof the 1H6, 38A8, 12B2, 38F6, 2A2, 36A8, 4B11, 34D10, 28F4, 23C10, 28C2,5C4, 9D6, and 18F7 antibodies, indicating the location of thecomplementarity determining regions (CDR). The location of each CDR(CDR1, CDR2, and CDR3) according to the EU index is indicated. Thelocation of identical, conserved and partially conserved amino acidresidues is indicated below the alignment.

FIG. 7 shows a ClustalX multiple sequence alignment of the VL sequencesof the 28C2, 9D6, 1H6, 38A8, 12B2, 18F7, 28F4, 34D10, 36A8, and 2A2antibodies, indicating the location of CDR1, CDR2, and CDR3 according tothe EU index. The location of identical, conserved and partiallyconserved amino acid residues is indicated below the alignment.

FIG. 8 shows percentage identity matrices corresponding to the sequencesincluded in the ClustalX multiple sequence alignments shown in FIG. 7(top matrix) and FIG. 6 (bottom matrix).

FIG. 9 shows ClustalX multiple sequence alignments corresponding to theVH sequences in FIG. 2 clustered according to their specificity for theα or β subunit of GPIIb/IIIa.

FIG. 10 shows ClustalX multiple sequence alignments corresponding to theVL sequences in FIG. 3 clustered according to their specificity for theα or β subunit of GPIIb/IIIa.

FIG. 11 shows ClustalX multiple sequence alignments corresponding to theVH sequences in FIG. 2 clustered according to their ability to competewith fibrinogen for binding to GPIIb/IIIa.

FIG. 12 shows ClustalX multiple sequence alignments corresponding to theVL sequences in FIG. 2 clustered according to their ability to competewith fibrinogen for binding to GPIIb/IIIa.

FIGS. 13A and 13B are schematic representations of an anti-GPIIb/IIIaantibody in scFv format recombinantly fused to the FVIIa clotting factorheavy chain (FIG. 13A) or light chain (FIG. 13B) for platelet targeting.

FIG. 14 is a diagram summarizing the experimental method used to studythe binding kinetics and affinity of a platelet-targeted rFVIIa chimericmolecule for GPIIb/IIIa. GPIIb/IIIa constructs were doubly labeled withbiotin and the HPC4 purification tag and incorporated into GPIIb/IIIananodiscs. Binding of the platelet-targeted rFVIIa to the nanodiscs wasstudied using Surface Plasmon Resonance (SPR).

FIGS. 15A-15B show single cycle steady-state binding of aplatelet-targeted rFVIIa chimeric molecule to GPIIb/IIIa nanodiscsmeasured by SPR. FIG. 15A shows the binding as a function of time, andFIG. 15B shows the binding as a function of concentration.

FIGS. 16A-16B show ROTEM assay results corresponding to rFVIIa comparedto FVII-189 (a platelet-targeted FVIIa chimeric molecule where thetargeting moiety is an scFv derived from the 34D10 antibody) (FIG. 16A),and to rFVIIa compared to FVII-206 (a platelet-targeted FVIIa chimericmolecule where the targeting moiety is an scFv derived from the 12B2antibody) (FIG. 16B).

FIGS. 17A-17B show ROTEM assay results corresponding to rFVIIa comparedto FVII-204 (a platelet-targeted FVIIa chimeric molecule where thetargeting moiety is an scFv derived from the 38A8 antibody) (FIG. 17A),and to rFVIIa compared to FVII-163 (a platelet-targeted FVIIa chimericmolecule where the targeting moiety is an scFv derived from the PDG13antibody) and FVII-189 (FIG. 17B).

FIG. 18 shows the result of flow cytometry based platelet-bindingassays, in which the median fluorescence intensity (MFI) was plottedagainst the concentration of the chimeric molecules spiked in blood. MFIrepresented the relative concentration of the chimeric molecules thatbound to human platelets.

FIG. 19 shows flow cytometry profiles of the activation of gel-purifiedplatelets. Platelets were non-activated, activated after incubation withan agonist, or incubated with chimeric molecules FVII-189, FVII-206, andFVII-130 (a platelet-targeted FVII comprising an scFv derived from theAP3 antibody and an Fc moiety).

FIGS. 20A-20B show ADP-induced aggregation of platelet-rich plasma inthe presence of chimeric molecules FVII-189 (FIG. 20A) or FVII-206 (FIG.20B). In both cases, ADP and REOPRO® (Abciximab) were used as controls.

FIG. 21 shows human platelet clearance from NSG mice afteradministration of the chimeric molecules FVII-189, FVII-206, FVII204, orFVII-130. The NSG mice had been transfused with human platelets beforetreating with the chimeric molecules.

FIG. 22 shows possible configuration for chimeric molecules comprisingone or two heterologous moieties (H1 and/or H2) and scFv moietiesderived for GPIIb/IIIa-specific antibodies.

FIGS. 23A-23F show possible configurations for chimeric moleculescomprising the heavy and light chains of a clotting factor (e.g., FVII),an scFv targeting moiety (e.g., derived for GPIIb/IIIa-specificantibodies), a heterologous moiety (e.g., a half-life extending moiety),and at least one optional linker.

DETAILED DESCRIPTION

The clotting factor FVIIa has low affinity for platelets, the site ofaction for clot formation. Thus, a possible approach to increaseactivity of FVIIa is to target the clotting to platelet receptors viatargeting moieties (e.g., Fab or scFv), which can increase the affinityof FVIIa for platelets thereby boosting activity. Multiple receptors areexpressed exclusively on platelet. For example, GPIIb/IIIa (αIIbβ3) isan integrin specific to platelets which expressed at high levels.Activated GPIIb/IIIa receptor binds fibrinogen and modulates plateletaggregation. Upon activation, the GPIIb/IIIa receptors change from abent low ligand affinity conformation to an extended high ligandaffinity conformation. Thus, targeting strategies can be directed eitherthe active or the non-active conformations of the receptor.

The present disclosure provides antibodies against GPIIb/IIIa that arecapable of targeting the non-active form of the receptor. The GPIIb/IIIaantibodies and antigen-binding molecules derived from these antibodiescan be non-activating, and be used, for example, to target therapeuticmolecules (e.g., clotting factors or other molecules capable of having apharmacological effect in platelets) to the platelet surface. Inaddition to their use as targeting moieties, these antibodies andantigen-binding molecules thereof can be used for diagnostics, forexample, by conjugation to a detectable label.

In some embodiments, the GPIIb/IIIa antibodies and antigen-bindingmolecules thereof can be activating, for example, (i) by directlyactivating the GPIIb/IIIa receptors through binding to the α and/or βsubunits of the receptor, (ii) by indirectly activating the GPIIb/IIIavia simultaneous binding to the α and/or β subunits of the receptor andto an Fc receptor (e.g., an FcγII receptor), or (iii) by interferingwith the interaction between the GPIIb/IIIa receptor and fibrinogen.

In addition, the present invention relates to chimeric moleculescomprising the GPIIb/IIIa antibodies and antigen-binding moleculesthereof disclosed herein as targeting moieties, and one or moreheterologous moieties. For example, a heterologous moiety comprising atherapeutic molecule (for example, a procoagulant molecule such as aclotting factor), and optionally a second heterologous moietycomprising, for example, a PK enhancing moiety (i.e., a molecule whichcan improve various pharmacokinetic properties, e.g., half-life).

Exemplary GPIIb/IIIa antibodies and antigen-binding molecules thereof,as well as exemplary constructs (chimeric molecules) comprising suchantibodies and antigen-binding molecules thereof (e.g., scFv or F(ab))are illustrated in the instant description and figures. In someembodiments, the invention pertains to chimeric molecules having thestructures set forth, for example, in FIGS. 22 and 23. In otherembodiments, the invention pertains to polynucleotide encoding chimericmolecule constructs disclosed herein.

In order to provide a clear understanding of the specification andclaims, the following definitions are provided below.

I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleotide sequence,” is understood torepresent one or more nucleotide sequences. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about.” In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges. Asused herein, the terms “about” and “approximately” when referring to anumerical value shall have their plain and ordinary meanings to oneskilled in the art relevant to the range or element at issue.

The amount of broadening from the strict numerical boundary depends uponmany factors. For example, some of the factors to be considered caninclude the criticality of the element and/or the effect a given amountof variation will have on the performance of the claimed subject matter,as well as other considerations known to those of skill in the art.Thus, as a general matter, “about” or “approximately” broaden thenumerical value. For example, in some cases, “about” or “approximately”can mean±5%, or ±10%, depending on the relevant technology. Also, thedisclosure of ranges is intended as a continuous range including everyvalue between the minimum and maximum values recited.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various embodimentsof the disclosure, which can be by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety. Amino acidsare referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “amino acid chain,” or any other term used to refer to achain or chains of two or more amino acids, are included within thedefinition of “polypeptide,” and the term “polypeptide” can be usedinstead of, or interchangeably with any of these terms.

As used herein the term “protein” is intended to encompass a moleculecomprised of one or more polypeptides, which can in some instances beassociated by bonds other than amide bonds.

Polypeptides can be either monomers or multimers. For example, in oneembodiment, an antibody, an antigen-binding molecule thereof, or achimeric molecule of the invention can be a dimeric polypeptide. Adimeric antibody, an antigen-binding molecule thereof can comprise twopolypeptide chains or can consist of one polypeptide chain (e.g., in thecase of an scFc molecule). In one embodiment, the dimers can be ahomodimer, comprising two identical monomeric subunits or polypeptides(e.g., two identical Fc moieties or two identical biologically activemoieties). In another embodiment, the dimers are heterodimers,comprising two non-identical monomeric subunits or polypeptides (e.g.,comprising two different clotting factors or portions thereof or oneclotting factor only). See, e.g., U.S. Pat. No. 7,404,956, incorporatedherein by reference.

The terms “polypeptide” and “protein” are also intended to refer to theproducts of post-expression modifications, including without limitationglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, ormodification by non-naturally occurring amino acids. A polypeptide orprotein can be derived from a natural biological source or produced byrecombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

A polypeptide which is “isolated” is a polypeptide which is in a formnot found in nature. Isolated polypeptides include those which have beenpurified to a degree that they are no longer in a form in which they arefound in nature. In some embodiments, a polypeptide which is isolated issubstantially pure.

“Derivatives” of GPIIb/IIIa antibodies, antigen-binding moleculesthereof, or chimeric molecules of the invention are polypeptides orproteins which have been altered so as to exhibit additional featuresnot found on the native polypeptide or protein. Also included as“derivatives” are those peptides that contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Apolypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide. In oneembodiment, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least about 10 to about 20 amino acids, at least about 20to about 30 amino acids, or at least about 30 to about 50 amino acids,or which is otherwise identifiable to one of ordinary skill in the artas having its origin in the sequence.

Polypeptides that are “variants” of another polypeptide can have one ormore mutations relative to the starting polypeptide, e.g., one or moreamino acid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions. In one embodiment, the polypeptide comprises an amino acidsequence which is not naturally occurring. Such variants necessarilyhave less than 100% sequence identity or similarity with the startingpolypeptide. In another embodiment, the variant will have an amino acidsequence from about 75% to less than 100% amino acid sequence identityor similarity with the amino acid sequence of the starting polypeptide,for example, from about 80% to less than 100%, from about 85% to lessthan 100%, from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%) and from about 95% to less than 100%, e.g.,over the length of the variant molecule. In one embodiment, there is oneamino acid difference between a starting polypeptide sequence and thesequence derived therefrom.

The term “fragment” when referring to GPIIb/IIIa antibodies,antigen-binding molecules thereof, chimeric molecules of the invention,or clotting factors refers to any polypeptides or proteins which retainat least some of the properties of the reference polypeptide or protein.Fragments of polypeptides include proteolytic fragments, as well asdeletion fragments.

The term “sequence” as used to refer to a protein sequence, a peptidesequence, a polypeptide sequence, or an amino acid sequence means alinear representation of the amino acid constituents in the polypeptidein an amino-terminal to carboxyl-terminal direction in which residuesthat neighbor each other in the representation are contiguous in theprimary structure of the polypeptide.

The term “amino acid” includes alanine (Ala or A); arginine (Arg or R);asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C);glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G);histidine (His or H); isoleucine (Ile or I): leucine (Leu or L); lysine(Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline(Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp orW); tyrosine (Tyr or Y); and valine (Val or V).

Non-traditional amino acids are also within the scope of the inventionand include norleucine, omithine, norvaline, homoserine, and other aminoacid residue analogues such as those described in Ellman et al. Meth.Enzym. 202:301-336 (1991). To generate such non-naturally occurringamino acid residues, the procedures of Noren et al. Science 244:182(1989) and Ellman et al., supra, can be used. Briefly, these proceduresinvolve chemically activating a suppressor tRNA with a non-naturallyoccurring amino acid residue followed by in vitro transcription andtranslation of the RNA. Introduction of the non-traditional amino acidcan also be achieved using peptide chemistries known in the art. As usedherein, the term “polar amino acid” includes amino acids that have netzero charge, but have non-zero partial charges in different portions oftheir side chains (e.g., M, F, W, S, Y, N, Q, and C). These amino acidscan participate in hydrophobic interactions and electrostaticinteractions. As used herein, the term “charged amino acid” includesamino acids that can have non-zero net charge on their side chains (e.g.R, K, H, E, and D). These amino acids can participate in hydrophobicinteractions and electrostatic interactions.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, the presentlarger “peptide insertions”, can be made, e.g. insertion of about threeto about five or even up to about ten, fifteen, or twenty amino acidresidues. The inserted residue(s) can be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., Lys, Arg,and His), acidic side chains (e.g., Asp and Glu), uncharged polar sidechains (e.g., Gly, Asn, Gnl, Ser, Thr, Tyr, and Cys), nonpolar sidechains (e.g., Ala, Val, Leu, Ile, Pro, Phe, Met, and Trp), beta-branchedside chains (e.g., Thr, Val, and Ile) and aromatic side chains (e.g.,Tyr, Phe, Trp, and His). Thus, if an amino acid in a polypeptide isreplaced with another amino acid from the same side chain family, thesubstitution is considered to be conservative. In another embodiment, astring of amino acids can be conservatively replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members.

Non-conservative substitutions include those in which (i) a residuehaving an electropositive side chain (e.g., Arg, His, or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp),(ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by,a hydrophobic residue (e.g., Ala, Leu, He, Phe, or Val), (iii) acysteine or proline is substituted for, or by, any other residue, or(iv) a residue having a bulky hydrophobic or aromatic side chain (e.g.,Val, He, Phe, or Trp) is substituted for, or by, one having a smallerside chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

The term “percent sequence identity” between two polynucleotide orpolypeptide sequences refers to the number of identical matchedpositions shared by the sequences over a comparison window, taking intoaccount additions or deletions (i.e., gaps) that must be introduced foroptimal alignment of the two sequences. A matched position is anyposition where an identical nucleotide or amino acid is presented inboth the target and reference sequence. Gaps presented in the targetsequence are not counted since gaps are not nucleotides or amino acids.Likewise, gaps presented in the reference sequence are not counted sincetarget sequence nucleotides or amino acids are counted, not nucleotidesor amino acids from the reference sequence.

The percentage of sequence identity is calculated by determining thenumber of positions at which the identical amino acid residue or nucleicacid base occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. The comparison ofsequences and determination of percent sequence identity between twosequences can be accomplished using readily available software both foronline use and for download. Suitable software programs are availablefrom various sources, and for alignment of both protein and nucleotidesequences.

One suitable program to determine percent sequence identity is bl2seq,part of the BLAST suite of program available from the U.S. government'sNational Center for Biotechnology Information BLAST web site(blast.ncbi.nlm.nih.gov). B12seq performs a comparison between twosequences using either the BLASTN or BLASTP algorithm. BLASTN is used tocompare nucleic acid sequences, while BLASTP is used to compare aminoacid sequences. Other suitable programs are, e.g., Needle, Stretcher,Water, or Matcher, part of the EMBOSS suite of bioinformatics programsand also available from the European Bioinformatics Institute (EBI) atwww.ebi.ac.uk/Tools/psa.

Different regions within a single polynucleotide or polypeptide targetsequence that aligns with a polynucleotide or polypeptide referencesequence can each have their own percent sequence identity. It is notedthat the percent sequence identity value is rounded to the nearesttenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to80.2. It also is noted that the length value will always be an integer.

In certain embodiments, the percentage identity “X” of a first aminoacid sequence to a second sequence amino acid is calculated as100×(Y/Z), where Y is the number of amino acid residues scored asidentical matches in the alignment of the first and second sequences (asaligned by visual inspection or a particular sequence alignment program)and Z is the total number of residues in the second sequence. If thelength of a first sequence is longer than the second sequence, thepercent identity of the first sequence to the second sequence will behigher than the percent identity of the second sequence to the firstsequence.

One skilled in the art will appreciate that the generation of a sequencealignment for the calculation of a percent sequence identity is notlimited to binary sequence-sequence comparisons exclusively driven byprimary sequence data. Sequence alignments can be derived from multiplesequence alignments. One suitable program to generate multiple sequencealignments is ClustalW2, available from www.clustal.org (ClustalX is aversion of the ClustalW2 program ported to the Windows environment).Another suitable program is MUSCLE, available fromwww.drive5.com/muscle. ClustalW2 and MUSCLE are alternatively available,e.g., from the EBI.

It will also be appreciated that sequence alignments can be generated byintegrating sequence data with data from heterogeneous sources such asstructural data (e.g., crystallographic protein structures), functionaldata (e.g., location of mutations), or phylogenetic data. A suitableprogram that integrates heterogeneous data to generate a multiplesequence alignment is T-Coffee, available at www.tcoffee.org, andalternatively available, e.g., from the EBI. It will also be appreciatedthat the final alignment used to calculate percent sequence identity canbe curated either automatically or manually.

In one embodiment, the antibodies and antigen-binding molecules thereof,as well as the chimeric molecules of the invention can comprise an aminoacid sequence derived from a human protein sequence. However, theantibodies and antigen-binding molecules thereof, as well as thechimeric molecules of the invention can comprise one or more amino acidsfrom another mammalian species. In a particular embodiment, theantibodies and antigen-binding molecules thereof, as well as thechimeric molecules of the invention are not immunogenic.

As used herein, the terms “linked,” “fused”, or “fusion” refer tolinkage via a peptide bonds (e.g., genetic fusion), chemicalconjugation, or other means known in the art. For example, one way inwhich molecules or moieties can be linked employs peptide linkers whichlink the molecules or moieties via peptide bonds. The terms “geneticallyfused,” “genetically linked,” or “genetic fusion” are usedinterchangeably and refer to the co-linear, covalent linkage orattachment of two or more proteins, polypeptides, or fragments thereofvia their individual peptide backbones, through genetic expression of asingle polynucleotide molecule encoding those proteins, polypeptides, orfragments. Such genetic fusion results in the expression of a singlecontiguous genetic sequence.

Preferred genetic fusions are in frame, i.e., two or more open readingframes (ORFs) are fused to form a continuous longer ORF, in a mannerthat maintains the correct reading frame of the original ORFs. Thus, theresulting recombinant fusion protein is a single polypeptide containingtwo or more protein segments that correspond to polypeptides encoded bythe original ORFs (which segments are not normally so joined in nature).In this case, the single polypeptide is cleaved during processing toyield dimeric molecules comprising two polypeptide chains.

As used herein the term “associated with” refers to a covalent ornon-covalent bond formed between a first amino acid chain and a secondamino acid chain. In one embodiment, the term “associated with” means acovalent, non-peptide bond or a non-covalent bond. In anotherembodiment, the term “associated with” refers to a covalent, non-peptidebond or a non-covalent bond that is not chemically crosslinked. Inanother embodiment, it means a covalent bond except a peptide bond. Insome embodiments this association is indicated by a colon, i.e., (:).For example, when representing the structure of the clotting factor,“CF_(H):CF_(L)” refers to a dimer comprising a heavy chain of a clottingfactor (CF_(H)) disulfide bonded to a light chain of a clotting factor(CF_(L)) in a N-terminus to C-terminus orientation.

Examples of covalent bonds include, but are not limited to, a peptidebond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, api bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond,a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruplebond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation,aromaticity, hapticity, or antibonding. Non-limiting examples ofnon-covalent bond include an ionic bond (e.g., cation-pi bond or saltbond), a metal bond, an hydrogen bond (e.g., dihydrogen bond, dihydrogencomplex, low-barrier hydrogen bond, or symmetric hydrogen bond), van derWalls force, London dispersion force, a mechanical bond, a halogen bond,aurophilicity, intercalation, stacking, entropic force, or chemicalpolarity.

As used herein, the terms “chemically crosslinked” and “conjugated” areused interchangeably and refer to chemically linking by covalent bondsbetween acid side chains of amino acids, either directly or via alinker, e.g., a peptide linker. Chemical crosslinking does not includeintramolecular or intermolecular disulfide bonds between Fc moieties ofa dimeric Fc region, or non-engineered disulfide bonds between an aminoacid of the activatable clotting factor and an amino acid of theenhancer moiety. Chemical crosslinking generally takes place by additionof a cross-linking agent, e.g., a heterobifunctional crosslinking agent.Examples of chemical crosslinking includes one or more photo-reactivebonds by chemically connecting photo-Ile, photo-Met, and photo-Leu (see,Suchanek et al., (2005) Nature Methods, 2: 261-267).

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein (e.g., the GPIIb/IIIareceptor, a subunit thereof, or the receptor complex), polypeptide,peptide, carbohydrate, polynucleotide, lipid, or combinations of theforegoing through at least one antigen recognition site within thevariable region of the immunoglobulin molecule.

A typical antibody comprises at least two heavy (HC) chains and twolight (LC) chains interconnected by disulfide bonds. Each heavy chain iscomprised of a “heavy chain variable region” or “heavy chain variabledomain” (abbreviated herein as VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2,and CH3. Each light chain is comprised of a “light chain variableregion” or “light chain variable domain” (abbreviated herein as VL) anda light chain constant region. The light chain constant region iscomprised of one domain, Cl. The VH and VL regions can be furthersubdivided into regions of hypervariablity, termed ComplementarityDetermining Regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FW).

Each VH and VL region is composed of three CDRs and four FWs, arrangedfrom amino-terminus to carboxy-terminus in the following order: FW1,CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy andlight chains contain a binding domain that interacts with an antigen. Asused herein, the term “antibody” encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv),minibodies, multispecific antibodies such as bispecific antibodiesgenerated from at least two intact antibodies, chimeric antibodies,humanized antibodies, human antibodies, fusion proteins comprising anantigen determination portion of an antibody, and any other modifiedimmunoglobulin molecule comprising an antigen recognition site so longas the antibodies exhibit the desired biological activity. Thus, theterm “antibody” includes whole antibodies and any antigen-bindingfragment or single chains thereof. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

There are at least two techniques for determining the location of CDRs:(1) an approach based on cross-species sequence variability (i.e., Kabatet al. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-lazikaniet al. (1997) J. Molec. Biol. 273:927-948)). In addition, combinationsof these two approaches are sometimes used in the art to determine CDRs.

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence can containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FW or CDR of the variable domain. For example, a heavychain variable domain can include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavychain FW residue 82.

The Kabat numbering of residues can be determined for a given antibodyby alignment at regions of homology of the sequence of the antibody witha “standard” Kabat numbered sequence. Chothia refers instead to thelocation of the structural loops (Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34). The AbMhypervariable regions represent a compromise between the Kabat CDRs andChothia structural loops, and are used by Oxford Molecular's AbMantibody modeling software.

IMGT (ImMunoGeneTics) also provides a numbering system for theimmunoglobulin variable regions, including the CDRs. See e.g., Lefranc,M. P. et al., Dev. Comp. Immunol. 27: 55-77(2003). The IMGT numberingsystem was based on an alignment of more than 5,000 sequences,structural data, and characterization of hypervariable loops and allowsfor easy comparison of the variable and CDR regions for all species.According to the IMGT numbering schema VH-CDR1 is at positions 26 to 35,VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102,VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, andVL-CDR3 is at positions 89 to 97.

As used throughout the specification the VH CDR sequences describedherein correspond to the classical Kabat numbering locations, namelyKabat VH-CDR1 is at positions 31-35, VH-CDR2 is a positions 50-65, andVH-CDR3 is at positions 95-102. VL-CDR1, VL-CDR2, and VL-CDR3 alsocorrespond to classical Kabat numbering locations, namely positions14-24, 50-56 and 89-97, respectively.

The term “consensus sequence,” as used herein with respect to a CDR inthe light chain (VL) or heavy chain (VH) variable regions, refers to acomposite or genericized amino acid sequence defined based oninformation as to which amino acid residues are present at a givenposition based in multiple sequence alignments. Thus, in a “consensussequence” for a VL or VH chain CDR1, CDR2, or CDR3, certain amino acidpositions are occupied by one of multiple possible amino acid residuesat that position. For example, if an arginine (R) or a serine (S) occurat a particular position X, then that particular position within theconsensus sequence can be either arginine or serine (R or S). Suchoccurrence would be represented, for example, as_(N)-Z₁Z₂X_(n)Z_(t-1)Z_(t-C), where Z_(1>t) are invariant amino acids inthe multiple sequence alignment, X represent a position occupied byvariant amino acids (e.g., R or S), and the subindex n is an ordinal. Asused herein, referring to a polypeptide sequence as consisting of orcomprising a consensus sequence means that the polypeptide sequenceconsists of or comprises one of the of multiple possible amino acidsequences represented by the consensus sequence.

The term “antigen binding fragment” refers to a portion of an intactantibody and refers to the antigenic determining variable regions of anintact antibody. It is known in the art that the antigen bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

The term “Fab” refers to an antibody fragment that is essentiallyequivalent to that obtained by digestion of immunoglobulin (typicallyIgG) with the enzyme papain. The heavy chain segment of the Fab fragmentis the Fd piece. Such fragments can be enzymatically or chemicallyproduced by fragmentation of an intact antibody, recombinantly producedfrom a gene encoding the partial antibody sequence, or it can be whollyor partially synthetically produced.

The term “Fab′” refers to an antibody fragment that is essentiallyequivalent to that obtained by reduction of the disulfide bridge orbridges joining the two heavy chain pieces in the F(ab′)2 fragment. Suchfragments can be enzymatically or chemically produced by fragmentationof an intact antibody, recombinantly produced from a gene encoding thepartial antibody sequence, or it can be wholly or partiallysynthetically produced.

The term “F(ab′)2” refers to an antibody fragment that is essentiallyequivalent to a fragment obtained by digestion of an immunoglobulin(typically IgG) with the enzyme pepsin at pH 4.0-4.5. Such fragments canbe enzymatically or chemically produced by fragmentation of an intactantibody, recombinantly produced from a gene encoding the partialantibody sequence, or it can be wholly or partially syntheticallyproduced.

The term “Fv” refers to an antibody fragment that consists of one NH andone N domain held together by noncovalent interactions.

The term “monoclonal antibody” refers to a homogeneous antibodypopulation involved in the highly specific recognition and binding of asingle antigenic determinant, or epitope. This is in contrast topolyclonal antibodies that typically include different antibodiesdirected against different antigenic determinants. The term “monoclonalantibody” encompasses both intact and full-length monoclonal antibodiesas well as antibody fragments (such as Fab, Fab′, F(ab′)2, or Fv),single chain (scFv) mutants, fusion proteins comprising an antibodyportion, and any other modified immunoglobulin molecule comprising anantigen recognition site. Furthermore, “monoclonal antibody” refers tosuch antibodies made in any number of ways including, but not limitedto, by hybridoma, phage selection, recombinant expression, andtransgenic animals.

The term “human antibody” refers to an antibody produced by a human oran antibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides. The term “humanized antibody” refers to an antibodyderived from a non-human (e.g., murine) immunoglobulin, which has beenengineered to contain minimal non-human (e.g., murine) sequences. Theterm “chimeric antibodies” refers to antibodies wherein the amino acidsequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc.) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

In one embodiment, an anti-GPIIa/IIIb antibody of the inventioncomprises an antibody variant. The term “antibody variant” or “modifiedantibody” includes an antibody which does not occur in nature and whichhas an amino acid sequence or amino acid side chain chemistry whichdiffers from that of a naturally-derived antibody by at least one aminoacid or amino acid modification as described herein. As used herein, theterm “antibody variant” includes synthetic forms of antibodies which arealtered such that they are not naturally occurring, e.g., antibodiesthat comprise at least two heavy chain portions but not two completeheavy chains (such as, domain deleted antibodies or minibodies);multispecific forms of antibodies (e.g., bispecific, trispecific, etc.)altered to bind to two or more different antigens or to differentepitopes on a single antigen; heavy chain molecules joined to scFvmolecules; single-chain antibodies; diabodies; triabodies; andantibodies with altered effector function and the like.

As used herein the term “scFv” or “scFv molecule” includes bindingmolecules which consist of one light chain variable domain (VL) or aportion thereof, and one heavy chain variable domain (VH) or a portionthereof, wherein each variable domain (or a portion thereof) is derivedfrom the same or different antibodies. Single chain Fv moleculespreferably comprise an scFv linker interposed between the VH domain andthe VL domain. Exemplary scFv molecules are known in the art and aredescribed, for example, in U.S. Pat. No. 5,892,019; Ho et al., Gene77:51 (1989); Bird et al., Science 242:423 (1988); Pantoliano et al.,Biochemistry 30:10117 (1991); Milenic et al., Cancer Research 51:6363(1991); Takkinen et al., Protein Engineering 4:837 (1991).

The term “scFv linker” as used herein refers to a moiety interposedbetween the VL and VH domains of the scFv. The scFv linkers preferablymaintain the scFv molecule in an antigen-binding conformation. In oneembodiment, a scFv linker comprises or consists of an scFv linkerpeptide. In certain embodiments, an scFv linker peptide comprises orconsists of a gly-ser peptide linker. In other embodiments, an scFvlinker comprises a disulfide bond.

As used herein, the term “antigen-binding molecule” refers to a moleculecomprising an anti-GPIIb/IIIa antibody fragment, variant, or derivativethereof, comprising at least one CDR from one or more of theanti-GPIIb/IIIa antibodies disclosed herein. In some embodiments, theantigen-binding molecule is a protein. In other embodiments, theantigen-binding molecule is a protein scaffold (e.g., a fibronectin typeIII domain) or non-protein scaffold comprising at least one CDR from oneof the anti-GPIIb/IIIa antibodies disclosed herein. In some embodiments,the antigen-binding molecule is an anti-GPIIb/IIIa antibody identifiedaccording to the methods disclosed herein, comprising at least one CDRidentical to one of the CDR sequences disclosed herein. The term“antigen-binding molecule” also encompasses any molecule comprising a VHand/or VL region from one or more of the anti-GPIIb/IIIa antibodiesdisclosed herein.

The term “polynucleotide” or “nucleotide” is intended to encompass asingular nucleic acid as well as plural nucleic acids and refers to anisolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA)or plasmid DNA (pDNA). In certain embodiments, a polynucleotidecomprises a conventional phosphodiester bond or a non-conventional bond(e.g., an amide bond, such as found in peptide nucleic acids (PNA)).

The term “nucleic acid” refers to any one or more nucleic acid segments,e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated”nucleic acid or polynucleotide is intended a nucleic acid molecule, DNAor RNA, which has been removed from its native environment. Examples ofan isolated polynucleotide include recombinant polynucleotidesmaintained in heterologous host cells or purified (partially orsubstantially) from other polynucleotides in a solution. Isolated RNAmolecules include in vivo or in vitro RNA transcripts of polynucleotidesof the present invention. Isolated polynucleotides or nucleic acidsaccording to the present invention further include such moleculesproduced synthetically. In addition, a polynucleotide or a nucleic acidcan include regulatory elements such as promoters, enhancers, ribosomebinding sites, or transcription termination signals.

As used herein, a “coding region” or “coding sequence” is a portion ofpolynucleotide which consists of codons translatable into amino acids.Although a “stop codon” (tag, tga, or taa) is typically not translatedinto an amino acid, it can be considered to be part of a coding region,but any flanking sequences, for example promoters, ribosome bindingsites, transcriptional terminators, introns, and the like, are not partof a coding region. The boundaries of a coding region are typicallydetermined by a start codon at the 5′ terminus, encoding the aminoterminus of the resultant polypeptide, and a translation stop codon atthe 3′terminus, encoding the carboxyl terminus of the resultingpolypeptide.

Two or more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. It follows, then, that a single vector can contain just asingle coding region, or comprise two or more coding regions, e.g., asingle vector can separately encode a binding domain-A and a bindingdomain-B as described below. In addition, a vector, polynucleotide, ornucleic acid of the invention can encode heterologous coding regions,either fused or unfused to a nucleic acid encoding a binding domain ofthe invention. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired polynucleotide in a cell. Asknown to those skilled in the art, such vectors can easily be selectedfrom plasmids, phages, viruses, or retroviruses. In general, vectorscompatible with the instant invention will comprise a selection marker,appropriate restriction sites to facilitate cloning of the desired geneand the ability to enter and/or replicate in eukaryotic or prokaryoticcells.

Numerous expression vector systems can be employed to produce theantibody, antigen-binding molecule thereof, or a chimeric molecule ofthe invention. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Additionally, cells which haveintegrated the DNA into their chromosomes can be selected by introducingone or more markers which allow selection of transfected host cells. Themarker can provide for prototrophy to an auxotrophic host, biocideresistance (e.g., antibiotics) or resistance to heavy metals such ascopper. The selectable marker gene can either be directly linked to theDNA sequences to be expressed, or introduced into the same cell bycotransformation. In one embodiment, an inducible expression system canbe employed. Additional elements can also be needed for optimalsynthesis of mRNA. These elements can include signal sequences, splicesignals, as well as transcriptional promoters, enhancers, andtermination signals. In one embodiment, a secretion signal, e.g., anyone of several well characterized bacterial leader peptides (e.g., pelB,phoA, or ompA), can be fused in-frame to the N terminus of a polypeptideof the invention to obtain optimal secretion of the polypeptide. (Lei etal. (1988), Nature, 331:543; Better et al. (1988) Science, 240:1041;Mullinax et al., (1990). PNAS, 87:8095).

Certain proteins secreted by mammalian cells are associated with asecretory signal peptide which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that signal peptides are generally fused to the N-terminus of thepolypeptide, and are cleaved from the complete or “full-length”polypeptide to produce a secreted or “mature” form of the polypeptide.In certain embodiments, a native signal peptide, e.g., an immunoglobulinheavy chain or light chain signal peptide is used, or a functionalderivative of that sequence that retains the ability to direct thesecretion of the polypeptide that is operably associated with it.Alternatively, a heterologous mammalian signal peptide, e.g., a humantissue plasminogen activator (TPA) or mouse β-glucuronidase signalpeptide, or a functional derivative thereof, can be used.

A “recombinant” polypeptide or protein refers to a polypeptide orprotein produced via recombinant DNA technology. Recombinantly producedpolypeptides and proteins expressed in host cells are consideredisolated for the purpose of the invention, as are native or recombinantpolypeptides which have been separated, fractionated, or partially orsubstantially purified by any suitable technique.

The term “host cell” refers to a cell that has been transformed with avector constructed using recombinant DNA techniques and encoding atleast one heterologous gene. In descriptions of processes for isolationof proteins from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of protein unless it isclearly specified otherwise. In other words, recovery of protein fromthe “cells” can mean either from spun down whole cells, or from the cellculture containing both the medium and the suspended cells. The hostcell line used for protein expression is most preferably of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO cell line, BHK cellline, HEK cell line, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFRminus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS(a derivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), PerC6 cells), HAK (hamsterkidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma),BFA-1clBPT (bovine endothelial cells), and RAJI (human lymphocyte). Hostcell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

II. Anti-GPIIb/IIIa Antibodies

The present invention provides antibodies and antigen-binding moleculesthereof that specifically bind to GPIIb/IIIa receptors located on thesurface of platelets. As used herein, the terms “GPIIb/IIIa” and“GPIIb/IIIa receptor” refer to glycoprotein IIb/IIIa (also known asintegrin αIIbβ3), an integrin complex found on platelets. Integrins arecomposed of two chains, an α subunit and a β subunit, which are heldtogether by noncovalent bonds in a calcium dependent manner. GPIIbconstitutes the α subunit, which comprises divalent cation bindingdomains, whereas GPIIIa is a pro typical β subunit (13). On eachcirculating platelet, there are 35,000 to 100,000 GPIIb/IIIa complexes;most are distributed on the platelet surface, with a smaller pool in aninternal reserve. The GPIIb/IIIa complex does not interact with itsplasma ligands until platelets have been activated by exogenous agonistssuch as ADP or thrombin. When this occurs, an inside-out signal isgenerated that results in a conformational change in the extracellularportion of the complex that renders the molecule capable of bindingfibrinogen and other ligands. See Uniprot entries P05106 (ITB3_HUMAN;GPIIIa: CD61; integrin beta-3; integrin 13) and P08514 (ITA2B_HUMAN;GPIIb; CD41; integrin alpha-2b; integrin all) as published in UniversalProtein Resource (Uniprot) database release 2013_05 (May 1, 2013), whichare incorporated by reference in their entireties.

The GPIIb/IIIa receptor is a target of several GpIIb/IIIa inhibitordrugs including abciximab (REOPRO®), eptifibatide (INTEGRILIN®), andtirofiban (AGGRASTAT®). GPIIb/IIIa inhibitors can be used duringpercutaneous coronary intervention (angioplasty with or withoutintracoronary stent placement) to preventing platelet aggregation andthrombus formation by inhibiting the GPIIb/IIIa receptor on the surfaceof the platelets. They can also be used to treat acute coronarysyndromes, without percutaneous coronary intervention, depending on TIMIrisk.

The terms “GPIIb/IIIa antibody,” “anti-GPIIb/IIIa antibody,”“anti-GPIIb/IIIa,” “antibody that binds to GPIIb/IIIa” and anygrammatical variations thereof refer to an antibody that is capable ofspecifically binding to the GPIIb/IIIa receptor with sufficient affinitysuch that the antibody is useful as a therapeutic agent or diagnosticreagent in targeting GPIIb/IIIa. The extent of binding of ananti-GPIIb/IIIa antibody disclosed herein to an unrelated,non-GPIIb/IIIa protein is less than about 10% of the binding of theantibody to GPIIb/IIIa as measured, e.g., by a radioimmunoassay (RIA),BIACORE™ (using recombinant GPIIb/IIIa as the analyte and antibody asthe ligand, or vice versa), or other binding assays known in the art. Incertain embodiments, an antibody that binds to GPIIb/IIIa has adissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤50 nM, ≤10 nM, ≤1 nM,≤0.1 nM, ≤10 pM, ≤1 pM, or ≤0.1 pM.

In some embodiments, the antibody or antigen-binding molecules thereofspecifically bind to a GPIIb/IIIa epitope, which comprises or overlapswith the GPIIb/IIIa binding epitope of an antibody selected from 34D10,12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6, 13C1,5C4, 23C10, 37C7, 28C2, 9D6, or 28F4 (see TABLE 1). In some embodiments,the antibody or antigen-binding molecules thereof specifically bind to aGPIIb/IIIa epitope, which is the same GPIIb/IIIa binding epitope of anantibody selected from 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8,21F10, 38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4 (seeTABLE 1). As used herein, the term “epitope” designates a specific aminoacid sequence, modified amino acid sequence, or protein secondary ortertiary structure which is specifically recognized by an antibody. Theterms “specifically recognizing,” “specifically recognizes,” and anygrammatical variants mean that the antibody or antigen-binding moleculethereof is capable of specifically interacting with and/or binding to atleast two, at least three, or at least four amino acids of an epitope,e.g., a GPIIb/IIIa epitope. Such binding can be exemplified by thespecificity of a “lock-and-key-principle.” Thus, specific motifs in theamino acid sequence of the antigen-binding domain the GPIIb/IIIaantibody or antigen-binding molecule thereof and the epitope bind toeach other as a result of their primary, secondary or tertiary structureas well as the result of secondary modifications of the structure.

In other embodiments, the antibody or antigen-binding molecule thereofof the present invention specifically binds to GPIIb/IIIa andcompetitively inhibits GPIIb/IIIa binding by an antibody selected from34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6,13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4 (see TABLE 1). In someembodiments, the antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope comprises at least one, atleast two, at least three, at least four, or at least fivecomplementarity determining regions (CDR) or variants thereof of anantibody selected from one or more of the 34D10, 12B2, 2A2, 35D1, 36A8,4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2,9D6, or 28F4 antibodies disclosed in TABLE 1. In other embodiments, theantibody or antigen-binding molecule thereof which specifically binds toa GPIIb/IIIa epitope comprises six CDRs or variants thereof of anantibody selected from one or more of the 34D10, 12B2, 2A2, 35D1, 36A8,4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2,9D6, or 28F4 antibodies disclosed herein. In some embodiments, CDRs areindependently selected from CDRs or variants thereof derived from the VHand/or VL region of one, two, three, four, or six antibodies selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4.

In certain embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VH-CDR1 of an antibody selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VH-CDR2 of an antibody selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to VH-CDR3 of an antibodyselected from 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10,38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VL-CDR1 of an antibody selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VL-CDR2 of an antibody selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4, and/or

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60,70, 80, 90, or 95% identical to VL-CDR3 of an antibody selected from34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6,13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4.

In certain embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VH-CDR1 of an antibody selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VH-CDR2 of an antibody selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4; and

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to VH-CDR3 of an antibodyselected from 4D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8,18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4.

In other embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VL-CDR1 of an antibody selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(ii) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VL-CDR2 of an antibody selectedfrom 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4, and

(iii) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60,70, 80, 90, or 95% identical to VL-CDR3 of an antibody selected from34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6,13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4.

In certain embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VH-CDR1 of an antibody selectedfrom 34D10, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, or 18F7;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VH-CDR2 of an antibody selectedfrom 34D10, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, or 18F7;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to VH-CDR3 of an antibodyselected from 34D10, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, or18F7;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VL-CDR1 of an antibody selectedfrom 34D10, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, or 18F7;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VL-CDR2 of an antibody selectedfrom 34D10, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, or 18F7,and/or

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60,70, 80, 90, or 95% identical to VL-CDR3 of an antibody selected from34D10, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, or 18F7.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VH-CDR1 of an antibody selectedfrom 12B2, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VH-CDR2 of an antibody selectedfrom 12B2, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to VH-CDR3 of an antibodyselected from 12B2, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VL-CDR1 of an antibody selectedfrom 12B2, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to VL-CDR2 of an antibody selectedfrom 12B2, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4, and/or

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60,70, 80, 90, or 95% identical to VL-CDR3 of an antibody selected from12B2, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 25, 31,37, 43, or 111;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS:26, 32,38, 44, or 112;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 27,33, 39, 45, or 113;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 28, 34,40, 117, or 114;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 29, 35,41, 118, or 115; and,

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60,70, 80, 90, or 95% identical to any one of SEQ ID NOS: 30, 36, 42, 119,or 116.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope, comprises:

(i) a VH-CDR1 comprising the consensus sequence XIYAMS wherein X₁represents any amino acid residue, e.g., an amino acid residue withuncharged polar side chain or nonpolar side chain, e.g., Thr (T), Ser(S), or Ala (A);

(ii) a VH-CDR2 comprising the consensus sequence SIX₂X₃GX₄X₅TYX₆X₇DSVKX₈ wherein X₂ represents any amino acid residue, e.g., an aminoacid residue with uncharged polar side chain, e.g., Ser (S) or Asn (N),X₃ represents any amino acid residue, e.g., an amino acid residue withuncharged polar side chain, e.g., Ser (S) or Gly (G), X₄ represents anyamino acid residue, e.g., an amino acid residue with uncharged polarside chain, e.g., Ser (S) or Gly (G), X₅ represents any amino acidresidue, e.g., an amino acid residue with uncharged polar side chain,e.g., Ser (S), Asn (N), or Thr (T), X₆ represents any amino acidresidue, e.g., an amino acid residue with aromatic side chain, e.g., Tyr(Y) or Phe (F), X₇ represents any amino acid residue, e.g., an aminoacid residue with nonpolar side chains, e.g., Leu (L) or Pro (P), and X₈represents any amino acid residue, e.g., an amino acid residue withbasic side chains or uncharged polar side chains, e.g., Gly (G) or Arg(R);

(iii) a VH-CDR3 comprising the consensus sequence GGDYGYAX₉DY, whereinX₉ represents any amino acid residue, e.g., an amino acid residue withnonpolar side chains, e.g., Leu (L) or Met (M);

(iv) a VL-CDR1 comprising the sequence RASSSVNYMY (SEQ ID NO: 28);

(v) a VL-CDR2 comprising the sequence YTSNLAP (SEQ ID NO: 29); and,

(vi) a VL-CDR3 comprising the sequence QQFSSSPWT (SEQ ID NO: 30).

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a VH-CDR1 sequence selected from SEQ ID NOS: 25, 31, 37, 43, or 111;

(ii) a VH-CDR2 sequence selected from SEQ ID NOS: 26, 32, 38, 44, or112;

(iii) a VH-CDR3 sequence selected from SEQ ID NOS: 27, 33, 39, 45, or113;

(iv) a VL-CDR1 sequence selected from SEQ ID NOS: 28, 34, 40, 117, or114;

(v) a VL-CDR2 sequence selected from SEQ ID NOS: 29, 35, 41, 118, or115; and,

(vi) a VL-CDR3 sequence selected from SEQ ID NOS: 30, 36, 42, 119, or116.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises an amino acidsequence at least about 80%, 85%, 90%, 95%, or 100% identical to any oneof SEQ ID NOS: 1, 3, 5, 7, or 97 and a VL region comprising an aminoacid sequence at least about 80%, 85%, 90%, 95%, or 100% identical toany one of SEQ ID NOS: 2, 4, 6, 99, or 98. In some embodiments, theantibody or antigen-binding molecule thereof comprises a VH regioncomprising the amino acid sequence of SEQ ID NO: 1 and a VL regioncomprising the amino acid sequence of SEQ ID NO: 2. In otherembodiments, the antibody or antigen-binding molecule thereof comprisesa VH region comprising the amino acid sequence of SEQ ID NO: 3 and a VLregion comprising the amino acid sequence of SEQ ID NO: 4. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH region comprising the amino acid sequence of SEQ ID NO: 5 and a VLregion comprising the amino acid sequence of SEQ ID NO: 6. In someembodiments, the antibody or antigen binding molecule thereof comprisesa VH region comprising the amino acid sequence of SEQ ID NO: 7 and a VLregion comprising the amino acid sequence of SEQ ID NO: 99. In someembodiments, the antibody or antigen binding molecule thereof comprisesa VH region comprising the amino acid sequence of SEQ ID NO: 97 and a VLregion comprising the amino acid sequence of SEQ ID NO: 98. In someembodiment, the anti-GPIIb/IIIa antibody or antigen-binding moleculethereof binds to a GPIIb/IIIa epitope located in the extracellulardomain of the alpha subunit of GPIIb/IIIa or to a binding site formed bythe extracellular domains of the GPIIb/IIIa complex. In someembodiments, the GPIIb/IIIa antibody or antigen-binding molecule thereofdoes not compete with fibrinogen for binding to GPIIb/IIIa.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 46, 52,120, or 126;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 47, 53,121, or 127;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 48,54, 122, or 128;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 49, 55,123, or 129;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 50, 56,124, or 130; and,

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NO: 51, 57,125, or 131.

In some embodiments. the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope, comprises:

(i) a VH-CDR1 comprising the sequence NYLIE (SEQ ID NO: 46);

(ii) a VH-CDR2 comprising the sequence VINPGSGGTNYNEKFKG (SEQ ID NO:47);

(iii) a VH-CDR3 comprising the sequence GRYEWYFDV (SEQ ID NO: 48);

(iv) a VL-CDR1 comprising the consensus sequence RASQDIX₁₀NYLN whereinX₁₀ represents any amino acid residue, e.g., an amino acid residue withuncharged polar side chain, e.g., Ser (S) or Thr (T);

(v) a VL-CDR2 comprising the sequence YTSRLHS (SEQ ID NO:50); and,

(vi) a VL-CDR3 comprising the sequence QQGYTLPYT (SEQ ID NO:51).

In some embodiments, the antibody or antigen-binding molecule thereofcomprises:

(i) a VH-CDR1 sequence selected from SEQ ID NOS: 46, 52, 120, or 126;

(ii) a VH-CDR2 sequence selected from SEQ ID NOS: 47, 53, 121, or 127;

(iii) a VH-CDR3 sequence selected from SEQ ID NOS: 48, 54, 122, or 128;

(iv) a VL-CDR1 sequence selected from SEQ ID NOS: 49, 55, 123, or 129;

(v) a VL-CDR2 sequence selected from SEQ ID NOS: 50, 56, 124, or 130;and,

(vi) a VL-CDR3 sequence selected from SEQ ID NOS: 51, 57, 125, or 131.

In some embodiments, the antibody or antigen-binding molecule thereof,which specifically binds to a GPIIb/IIIa epitope, comprises a VH regioncomprising an amino acid sequence at least about 80%, 85%, 90%, 95%, or100% identical to any one of SEQ ID NOS: 8, 10, 100, or 102 and a VLregion comprising an amino acid sequence at least about 80%, 85%, 90%,95%, or 100% identical to any one of SEQ ID NOS: 9, 11, 101, or 103. Insome embodiments, the antibody or antigen-binding molecule thereofcomprises a VH region comprising the amino acid sequence of SEQ ID NO: 8and a VL region comprising the amino acid sequence of SEQ ID NO: 9. Insome embodiments, the antibody or antigen-binding molecule thereofcomprises a VH region comprising the amino acid sequence of SEQ ID NO:10 and a VL region comprising the amino acid sequence of SEQ ID NO: 11.In some embodiments, the antibody or antigen-binding molecule thereofcomprises a VH region comprising the amino acid sequence of SEQ ID NO:100 and a VL region comprising the amino acid sequence of SEQ ID NO:101. In some embodiments, the antibody or antigen-binding moleculethereof comprises a VH region comprising the amino acid sequence of SEQID NO: 102 and a VL region comprising the amino acid sequence of SEQ IDNO: 103. In some embodiments, the antibody or antigen-binding moleculethereof binds to a GPIIb/IIIa epitope located in the extracellulardomain of the alpha subunit of GPIIb/IIIa or to a binding site formed bythe extracellular domains of the GPIIb/IIIa complex. In someembodiments, the antibody or antigen-binding molecule thereof competeswith fibrinogen for binding to GPIIb/IIIa.

In some embodiments. the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 58;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 59;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 60;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 61;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 62; and,

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 63.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a VH-CDR1 sequence comprising SEQ ID NO: 58;

(ii) a VH-CDR2 sequence comprising SEQ ID NO: 59;

(iii) a VH-CDR3 sequence comprising SEQ ID NO: 60;

(iv) a VL-CDR1 sequence comprising SEQ ID NO: 61;

(v) a VL-CDR2 sequence comprising SEQ ID NO: 62; and,

(vi) a VL-CDR3 sequence comprising SEQ ID NO: 63.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises a VH regioncomprising an amino acid sequence at least about 80%, 85%, 90%, 95%, or100% identical to SEQ ID NO: 12, and a VL region comprising an aminoacid sequence at least about 80%, 85%, 90%, 95%, or 100% identical toSEQ ID NO: 13. In some embodiments, the antibody or antigen-bindingmolecule thereof binds to a GPIIb/IIIa epitope is located in theextracellular domain of the alpha subunit of GPIIb/IIIa. In someembodiments, the antibody or antigen-binding molecule thereof competeswith fibrinogen for binding to GPIIb/IIIa.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 64, 70,or 135;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 65, 71,or 136;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 66,72, or 137;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 67, 132,or 138;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 68, 133,or 139; and,

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 69, 134,or 140.

In other embodiments, the invention includes an antibody orantigen-binding molecule thereof which specifically binds to aGPIIb/IIIa epitope, comprising (i) a VH-CDR1 comprising the sequenceSYWIE (SEQ ID NO: 64); (ii) a VH-CDR2 comprising the consensus sequenceEILPGX₁₄GX₁₅TKYNX₁₆KFKG (SEQ ID NO: 187), wherein X₁₄ represents anyamino acids, e.g., an amino acid residue with uncharged polar sidechain, e.g., Ser (S) or Thr (T), X₁₅ represents any amino acids, e.g.,an amino acid residue with uncharged polar side chains or beta-branchedside chains, e.g., Ile (I) or Tyr (Y), and X₁₆ represents any aminoacid, e.g., an amino acid residue with acidic side chains, e.g., Asp (D)or Glu (E); (iii) a VH-CDR3 comprising the sequence LISYYYAMDY (SEQ IDNO: 66);

(iv) a VL-CDR1 comprising the sequence RASQDISNYLN (SEQ ID NO: 67); (v)a VL-CDR2 comprising the sequence YTSRLHS (SEQ ID NO: 68); and, (vi) aVL-CDR3 comprising the sequence QQGNTLPPT (SEQ ID NO: 69).

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a VH-CDR1 sequence selected from SEQ ID NOS: 64, 70, or 135;

(ii) a VH-CDR2 sequence selected from SEQ ID NOS: 65, 71, or 136;

(iii) a VH-CDR3 sequence selected from SEQ ID NOS: 66, 72, or 137;

(iv) a VL-CDR1 sequence selected from SEQ ID NOS: 67, 132, or 138;

(v) a VL-CDR2 sequence selected from SEQ ID NOS: 68, 133, or 139; and,

(vi) a VL-CDR3 sequence selected from SEQ ID NOS: 69, 134, or 140.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises a VH regioncomprising an amino acid sequence at least about 80%, 85%, 90%, 95%, or100% identical to any one of SEQ ID NOS: 14, 16, or 105 and a VL regioncomprising an amino acid sequence at least about 80%, 85%, 90%, 95%, or100% identical to any one of SEQ ID NOS: 15, 104, or 106. In someembodiments, the antibody or antigen-binding molecule comprises a VHregion comprises the amino acid sequence of SEQ ID NO: 14 and a VLregion comprising the amino acid sequence of SEQ ID NO: 15. In someembodiments, the antibody or antigen-binding molecule thereof comprisesa VH region comprising the amino acid sequence of SEQ ID NO: 16 and a VLregion comprising the amino acid sequence of SEQ ID NO: 104. In someembodiments, the antibody or antigen-binding molecule comprises a VHregion comprising the amino acid sequence of SEQ ID NO: 105 and a VLregion comprises the amino acid sequence of SEQ ID NO: 106. In someembodiments, the antibody or antigen-binding molecule thereof binds to aGPIIb/IIIa epitope located in the extracellular domain of the betasubunit of GPIIb/IIIa. In some embodiments, the antibody orantigen-binding molecule thereof does not compete with fibrinogen forbinding to GPIIb/IIIa.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 73, 76,79, 85, or 147;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 74, 77,80, 86, or 148;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 75,78, 81, 87, or 149;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 141,144, 82, 88, or 150;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 142,145, 83, 89, or 151; and,

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to any one of SEQ ID NO: 143, 146,84, 90, or 152.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a VH-CDR1 comprising the consensus sequence TSGX₁₁GVG, wherein X₁₁represents any amino acid residue, e.g., an amino acid residue withnonpolar side chains, e.g., Met (M) or Leu (L);

(ii) a VH-CDR2 comprising the consensus sequence HIWWDDDKRYNPX₁₂LKS,wherein X₁₂ represents any amino acid residue, e.g., an amino acidresidue with nonpolar side chains or beta-branched side chains, e.g.,Ala (A) or Thr (T);

(iii) a VH-CDR3 comprising the consensus sequence SHYX₁₃GTFYFDX₁₄,wherein X₁₃ represents any amino acid residue, e.g., an amino acidresidue with uncharged polar side chain, e.g., Tyr (Y) or Asn (N), andX₁₄ represents any amino acid residue, e.g., an amino acid residue witharomatic side chain, e.g., Tyr (Y) or Phe (F);

(iv) a VL-CDR1 comprising the sequence RASKSISKYLA (SEQ ID NO: 82);

(v) a VL-CDR2 comprising the sequence SGSTLQS (SEQ ID NO: 83); and,

(vi) a VL-CDR3 comprising the sequence QQHIEYPWT (SEQ ID NO: 84).

In some embodiments, the antibody or antigen-binding molecule thereofaccording comprises:

(i) a VH-CDR1 sequence selected from SEQ ID NOS: 73, 76, 79, 85, or 147;

(ii) a VH-CDR2 sequence selected from SEQ ID NOS: 74, 77, 80, 86, or148;

(iii) a VH-CDR3 sequence selected from SEQ ID NOS: 75, 78, 81, 87, or149;

(iv) a VL-CDR1 sequence selected from SEQ ID NOS: 141, 144, 82, 88, or150;

(v) a VL-CDR2 sequence selected from SEQ ID NOS: 142, 145, 83, 89, or151; and,

(vi) a VL-CDR3 sequence selected from SEQ ID NOS: 143, 146, 84, 90, or152.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises a VH regioncomprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100%identical to any one of SEQ ID NOS: 17, 18, 19, 21, or 109 and a VLregion comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or100% identical to any one of SEQ ID NOS: 107, 108, 20, 22, or 110.

In some embodiments, the antibody or antigen-binding molecule thereofcomprises a VH region comprising the amino acid sequence of SEQ ID NO:17 and a VL region comprising the amino acid sequence of SEQ ID NO: 107.In other embodiments, the antibody or antigen-binding molecule thereofcomprises a VH region comprising the amino acid sequence of SEQ ID NO:18 and a VL region comprising the amino acid sequence of SEQ ID NO: 108.In some embodiments, the antibody or antigen-binding molecule thereofcomprises a VH region comprising the amino acid sequence of SEQ ID NO:109 and a VL region comprising the amino acid sequence of SEQ ID NO:110. In other embodiments, the antibody or antigen-binding moleculethereof comprises a VH region comprising the amino acid sequence of SEQID NO: 19 and a VL region comprising the amino acid sequence of SEQ IDNO: 20. In other embodiments, the antibody or antigen-binding moleculethereof comprises a VH region comprising the amino acid sequence of SEQID NO: 21 and a VL region comprising the amino acid sequence of SEQ IDNO: 22. In some embodiments, the antibody or antigen-binding moleculethereof binds to a GPIIb/IIIa epitope located in the extracellulardomain of the beta subunit of GPIIb/IIIa. In other embodiments, theantibody or antigen-binding molecule thereof competes with fibrinogenfor binding to GPIIb/IIIa.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a variable heavy chain CDR-1 (VH-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 91;

(ii) a variable heavy chain CDR-2 (VH-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 92;

(iii) a variable heavy chain CDR-3 (VH-CDR3) sequence at least about60%, 70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 93;

(iv) a variable light chain CDR-1 (VL-CDR1) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 94;

(v) a variable light chain CDR-2 (VL-CDR2) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 95; and,

(vi) a variable light chain CDR-3 (VL-CDR3) sequence at least about 60%,70%, 80%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises:

(i) a VH-CDR1 sequence comprising SEQ ID NO: 91;

(ii) a VH-CDR2 sequence comprising SEQ ID NO: 92;

(iii) a VH-CDR3 sequence comprising SEQ ID NO: 93;

(iv) a VL-CDR1 sequence comprising SEQ ID NO: 94;

(v) a VL-CDR2 sequence comprising SEQ ID NOS: 95; and,

(vi) a VL-CDR3 sequence comprising SEQ ID NOS: 96.

In some embodiments, the antibody or antigen-binding molecule thereofwhich specifically binds to a GPIIb/IIIa epitope comprises a VH regioncomprising an amino acid sequence at least about 80%, 85%, 90%, 95%, or100% identical to SEQ ID NO: 23 and a VL region comprising an amino acidsequence at least about 80%, 85%, 90%, 95%, or 100% identical to SEQ IDNO: 24. In some embodiments, the antibody or antigen-binding moleculethereof binds to a GPIIb/IIIa epitope located in the extracellulardomain of the “molecule thereof competes with fibrinogen for binding toGPIIb/IIIa.

In some embodiments, the anti-GPIIb/IIIa antibody or antigen-bindingmolecule thereof comprises or consists of (a) a single chain Fv(“scFv”); (b) a diabody; (c) a minibody; (d) a polypeptide chain of anantibody; (e) F(ab′)₂; or (f) F(ab).

In some embodiments, the anti-GPIIb/IIIa antibody or antigen-bindingmolecule thereof can be a targeting moiety. As used herein, the term“targeting moiety” refers to a moiety capable of interacting with atarget molecule (e.g., the GPIIb/IIIa receptor, or a molecule comprisingthe α and/or β subunits of the GPIIb/IIIa receptor). Targeting moietieshaving limited cross-reactivity are generally preferred. In certainembodiments, suitable targeting moieties include, for example, anymember of a specific binding pair, antibodies, monoclonal antibodies, orderivatives or analogs thereof, including without limitation: Fvfragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, camelized antibodies and antibodyfragments, humanized antibodies and antibody fragments, and multivalentversions of the foregoing; multivalent binding reagents includingwithout limitation: monospecific or bispecific antibodies, such asdisulfide stabilized Fv fragments, scFv tandems ((scFv) fragments),diabodies, tribodies or tetrabodies, which typically are covalentlylinked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; and other targeting moieties include forexample, aptamers, receptors, ligands, and fusion proteins. In someembodiments, an anti-GPIIb/IIIa antibody or antigen-binding moleculethereof disclosed herein can target GPIIb/IIIa located on the surface ofplatelets.

III. Chimeric Molecules

The present disclosure also provides “chimeric molecules” comprising,for example, at least one of the GPIIb/IIIa antibodies orantigen-binding molecules thereof disclosed herein which is fused and/orconjugated and/or otherwise associated with at least one heterologousmoiety. Thus, a chimeric molecule disclosed herein encompasses anymolecule comprising (i) a GPIIb/IIIa antibody or antigen-bindingmolecule thereof disclosed herein (e.g., a scFv derived, for example,from the 34D10 antibody or the 12B2 antibody), and (ii) at least oneheterologous moiety (e.g., a therapeutic moiety such as a clottingfactors, and optionally a half-life extending moiety). In someembodiments, a chimeric molecule is a chimeric protein, i.e., a chimericmolecule in which all its components (heterologous moieties and/orlinkers) are polypeptides. Other chimeric molecules can comprisenon-polypeptide heterologous moieties (e.g., PEG, lipids, carbohydrates,nucleic acids, small molecule therapeutic agents, radionuclides,fluorescent probes, etc.) and/or non-polypeptide linkers.

In some embodiments, a chimeric molecule comprises a first amino acidsequence derived from a first source, bonded, covalently ornon-covalently, to a second amino acid sequence derived from a secondsource, wherein the first and second source are not the same. A firstsource and a second source that are not the same can include twodifferent biological entities, or two different proteins from the samebiological entity, or a biological entity and a non-biological entity. Achimeric molecule can include for example, a protein derived from atleast 2 different biological sources. A biological source can includeany non-synthetically produced nucleic acid or amino acid sequence(e.g., a genomic or cDNA sequence, a plasmid or viral vector, a nativevirion or a mutant or analog, as further described herein, of any of theabove). A synthetic source can include a protein or nucleic acidsequence produced chemically and not by a biological system (e.g., solidphase synthesis of amino acid sequences). A chimeric molecule can alsoinclude a protein derived from at least 2 different synthetic sources ora protein derived from at least one biological source and at least onesynthetic source. A chimeric molecule can also comprise a first aminoacid sequence derived from a first source, covalently or non-covalentlylinked to a nucleic acid, derived from any source or a small organic orinorganic molecule derived from any source. The chimeric molecule canalso comprise a linker molecule between the first and second amino acidsequence or between the first amino acid sequence and the nucleic acid,or between the first amino acid sequence and the small organic orinorganic molecule.

As used herein the term “moiety” refers to a component part orconstituent of a chimeric molecule of the present invention. As usedherein, the term “heterologous moiety” refers to a moiety geneticallyfused, conjugated, and/or otherwise associated to a GPIIb/IIIa antibodyor antigen-binding molecule thereof. In some embodiments, the chimericmolecule has, for example, a formula:

(i) Tm-(L)-H or

(ii) H-(L)-Tm, wherein, H is a heterologous moiety; L is an optionallinker; and, Tm is an anti-GPIIb/IIIa antibody or antigen-bindingmolecule thereof disclosed herein.

In some embodiments, the chimeric molecule further comprises a secondheterologous moiety. Accordingly, in some embodiments, the chimericmolecule has a formula selected from:

(i) H1-(L1)-Tm-(L2)-H2;

(ii) H2-(L2)-Tm-(L1)-H1;

(iii) H1-(L1)-H2-(L2)-Tm;

(iv) H2-(L2)-H1-(L1)-Tm;

(v) Tm-(L1)-H1-(L2)-H2; or,

(vi) Tm-(L2)-H2-(L1)-H1; wherein, Tm is an anti-GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein; H1 is a firstheterologous moiety, H2 is a second heterologous moiety, L1 is a firstoptional linker, and L2 is a second optional linker.

In some embodiments, the first heterologous moiety and the secondheterologous moiety are the same. In other embodiments, the firstheterologous moiety and the second heterologous moiety are different. Insome embodiments, L1 and L2 are the same. In other embodiments, L1 andL2 are different.

The chimeric molecule formulas disclosed are oriented from N-terminus(left) to C-terminus (right). One skilled in the art would understandthat the chimeric molecule formulas disclosed herein are non-limitingexamples of chimeric molecules comprising the disclosed GPIIb/IIIaantibodies or antigen-binding molecules thereof. For example, theformulas can comprise further sequences at their N-terminal orC-terminal ends, or inserted between elements of the formula.Accordingly, a chimeric molecule can comprise, one, two, three, four,five, or more than five heterologous moieties. In some embodiments, thehyphen (-) in a formula indicates a peptide bond or one or more aminoacids. Exemplary chimeric molecules are presented in FIG. 22.

In some embodiments, a chimeric protein comprises a first polypeptidechain and a second polypeptide chain, which are associated with eachother. In some embodiments, the first polypeptide chain comprises alight chain of a clotting factor (e.g., FVII) and a heterologous moiety(e.g., a half-life extending moiety), and the second polypeptide chaincomprises a heavy chain of the clotting factor (e.g., FVII) and aGPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein. In other embodiments, the first polypeptide chain comprises alight chain of a clotting factor (e.g., FVII) and a GPIIb/IIIa antibodyor antigen-binding molecule thereof disclosed herein, and the secondpolypeptide chain comprises a heavy chain of the clotting factor (e.g.,FVII) and a heterologous moiety (e.g., a half-life extending moiety). Inyet another embodiment, the first polypeptide chain comprises a lightchain of a clotting factor (e.g., FVII) and the second polypeptide chaincomprises a heavy chain of the clotting factor (e.g., FVII), aGPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein, and a heterologous moiety (e.g., a half-life extending moiety).In some embodiments, the first polypeptide chain comprises a light chainof a clotting factor (e.g., FVII) and the second polypeptide chaincomprises a heavy chain of the clotting factor (e.g., FVII), aheterologous moiety (e.g., a half-life extending moiety), and aGPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein. In other embodiments, the first polypeptide chain comprises alight chain of a clotting factor (e.g., FVII), a heterologous moiety(e.g., a half-life extending moiety), and a GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein, and the secondpolypeptide chain comprises a heavy chain of the clotting factor (e.g.,FVII). In some embodiments, the first polypeptide chain comprises alight chain of a clotting factor (e.g., FVII), a GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein, and a heterologousmoiety (e.g., a half-life extending moiety), and the second polypeptidechain comprises a heavy chain of the clotting factor (e.g., FVII).

In some embodiments, the chimeric molecule comprises a formula:

(1) wherein the first polypeptide chain comprises CF_(L)-H or H-CF_(L)and the second polypeptide chain comprises CF_(H)-Tm or Tm-CF_(H);

(2) wherein the first polypeptide chain comprises CF_(L)-Tm or Tm-CF_(L)and the second polypeptide chain comprises CF_(H)-H or H-CF_(H);

(3) wherein the first polypeptide chain comprises CF_(L) and the secondpolypeptide chain comprises CF_(H)-Tm-H or H-Tm-CF_(H);

(4) wherein the first polypeptide chain comprises CF_(L) and the secondpolypeptide chain comprises CF_(H)-H-Tm or Tm-H-CF_(H);

(5) wherein the first polypeptide chain comprises CF_(L)-H-Tm orTm-H-CF_(L) and the second polypeptide chain comprises CF_(H); or

(6) wherein the first polypeptide chain comprises CF_(L)-Tm-H orH-Tm-CF_(L) and the second polypeptide chain comprises CF_(H);

wherein, CF_(L) is a light chain of a clotting factor (e.g., FVII);CF_(H) is a heavy chain of the clotting factor (e.g., FVII); Tm is ananti-GPIIb/IIIa antibody or antigen-binding molecule thereof, and H is aheterologous moiety (e.g., a half-life extending moiety). In someembodiments, the clotting factor is independently selected from thegroup consisting of FVII, FIX, FX, and any combinations thereof.

The instant disclosure also provides a chimeric molecule comprising afirst polypeptide chain and a second polypeptide chain, which areassociated with each other, (1) wherein the first polypeptide chaincomprises a light chain of a clotting factor (e.g., FVII, FIX, or FX),and a targeting moiety, which binds to a platelet, and the secondpolypeptide chain comprises a heavy chain of the clotting factor (e.g.,FVII, FIX, or FX) and a heterologous moiety (e.g., a half-life extendingmoiety); (2) wherein the first polypeptide chain comprises a light chainof a clotting factor (e.g., FVII) and a heterologous moiety (e.g., ahalf-life extending moiety), and the second polypeptide chain comprisesa heavy chain of the clotting factor (e.g., FVII, FIX, or FX) and atargeting moiety, which binds to a platelet; (3) wherein the firstpolypeptide chain comprises a light chain of a clotting factor (e.g.,FVII, FIX, or FX), a heterologous moiety (e.g., a half-life extendingmoiety), and a targeting moiety, which binds to a platelet, and thesecond polypeptide comprises a heavy chain of the clotting factor (e.g.,FVII, FIX, or FX); or (4) wherein the first polypeptide chain comprisesa light chain of a clotting factor (e.g., FVII, FIX, or FX), a targetingmoiety, which binds to a platelet, and a heterologous moiety (e.g., ahalf-life extending moiety) and the second polypeptide chain comprises aheavy chain of the clotting factor (e.g., FVII, FIX, or FX). In someembodiments, the clotting factor is FVII, FIX, or FX.

As used herein, the phrases “which binds to a platelet,” “binding to aplatelet,” and variants thereof generally refer to the specific bindingof (i) a GPIIb/IIIa antibody or antigen-binding molecule thereof or (ii)a chimeric molecule of the present disclosure to an antigenic site onthe surface of the platelet, e.g., an epitope on the extracellulardomains of the α and/or β subunits of the GPIIb/IIIa receptor. It wouldbe known to a person skilled in the art that GPIIb/IIIa is present intwo pools, a plasma membrane pool present in the platelet's restingstate and an internal pool of GPIIb/IIIa which is expressed uponplatelet activation. See, for example, Quinn et al., J. Pharmacol. Exp.Ther. 297:496-500 (2001). Accordingly, in some specific embodiments, andparticularly for diagnostic uses where the platelet's plasma membranecan be permeabilized, the binding of a GPIIb/IIIa antibody orantigen-binding molecule thereof to platelets, or the binding of achimeric molecule of the present disclosure to platelets can refer tobinding to the plasma membrane pool and/or to the internal pool ofGPIIb/IIIa.

In some embodiments, the chimeric molecule comprises a first polypeptidechain and a second polypeptide chain, which are associated with eachother, (1) wherein the first polypeptide chain comprises CF_(L)-H orH-CF_(L) and the second polypeptide chain comprises CF_(H)-Tm orTm-CF_(H); (2) wherein the first polypeptide chain comprises CF_(L)-Tmor Tm-CF_(L) and the second polypeptide chain comprises CF_(H)-H orH-CF_(H); (3) wherein the first polypeptide chain comprises CF_(L)-H-Tmor Tm-H-CF_(L) and the second polypeptide chain comprises CF_(H); or (4)wherein the first polypeptide chain comprises CF_(L)-Tm-H or H-Tm-CF_(L)and the second polypeptide chain comprises CF_(H); wherein, H is aheterologous moiety (e.g., a half-life extending moiety), CF_(H) is aheavy chain of a clotting factor (e.g., FVII), CF_(L) is a light chainof the clotting factor (e.g., FVII, FIX, or FX), Tm is a targetingmoiety which binds to a platelet, and L is an optional linker.

In some embodiments, the chimeric molecule comprises a formula selectedfrom (1) Tm-CF_(H):CF_(L)-H; (2) H-CF_(H):CF_(L)-Tm; (3)Tm-H-CF_(L):CF_(H); or (4) H-Tm-CF_(L): CF_(H); wherein, H is aheterologous moiety (e.g., a half-life extending moiety); CF_(H) is aheavy chain of a clotting factor (e.g., FVII, FIX, or FX); CF_(L) is alight chain of a clotting factor (e.g., FVII); Tm is a targeting moiety,which binds to a platelet; L is an optional linker; and: represents acovalent or non-covalent bond between CF_(H) and CF_(L) (e.g., adisulfide bond).

In some embodiments, the association between the first polypeptide chainand the second polypeptide chain in the chimeric molecule is a covalentbond or a non-covalent bond. Thus, in other embodiments, the associationbetween the first polypeptide chain and the second polypeptide chain inthe chimeric molecule is a covalent bond between the heavy chain and thelight chain of the clotting factor (e.g., FVII, FIX, or FX). Incontrast, in some other embodiments, the covalent bond is a disulfidebond.

The present disclosure also provides a chimeric molecule comprising asingle polypeptide chain, which comprises, from N terminus to Cterminus, (i) a light chain of a clotting factor (e.g., FVII, FIX, orFX), a heterologous moiety (e.g., a half-life extending moiety), aprotease cleavage site, a heavy chain of the clotting factor (e.g.,FVII, FIX, or FX), and a targeting moiety (e.g., a GPIIb/IIIa antibodyor antigen-binding molecule thereof) which binds to a platelet or (ii) alight chain of a clotting factor (e.g., FVII), a targeting moiety, whichbinds to a platelet, a protease cleavage site, a heavy chain of theclotting factor (e.g., FVII, FIX, or FX), and a heterologous moiety(e.g., a half-life extending moiety). In some embodiments, the clottingfactor is FVII. In other embodiments, the clotting factor is FIX or FX.In yet other embodiments, the clotting factor is FVII, FIX, or FX. Insome embodiments, the protease cleavage site is an intracellularprocessing site. In some embodiments, the intracellular processing siteis processed by a proprotein convertase. In some embodiments, theproprotein convertase is selected from the group consisting of PC5,PACE, PC7, and any combinations thereof.

In some embodiments, the targeting moiety in the chimeric molecule isselected from: an antibody or antigen binding molecule thereof, areceptor binding portion of a receptor, and a peptide. In someembodiments, the targeting moiety selectively binds to a restingplatelet or an activated platelet. In other embodiments, the targetingmoiety selectively binds to a target selected from the group consistingof: GP1ba (Uniprot: E7ES66; E7ES66_HUMAN), GPVI (Uniprot: Q9HCN6;GPVI_HUMAN), GPIX (Uniprot: P14770; GPIX_HUMAN), a nonactive form ofglycoprotein IIb/IIIa (“GPIIb/IIIa”), an active form of GPIIb/IIIa,P-selectin (Uniprot: Q14242; SELPL_HUMAN), GMP-33 (see, e.g., Damas etal., Thromb. Haemost. 86:887-93 (2001)), LAMP-1 (Uniprot: P11279;LAMP1_HUMAN), LAMP-2 (Uniprot: P13473; LAMP2_HUMAN), CD40L (Uniprot:P29965; CD40L_HUMAN), LOX-1 (Uniprot: P78380; OLR1_HUMAN), and anycombinations thereof. The above referenced Uniprot identifierscorrespond the entries published in the Universal Protein Resource(Uniprot) database release 2013_05 (May 1, 2013), and are incorporatedby reference in their entireties. In certain embodiments, the targetingmoiety comprises a GPIIb/IIIa antibody or antigen-binding moleculethereof. In specific embodiments, the GPIIb/IIIa antibody orantigen-binding molecule thereof is a GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein.

III.A Heterologous Moieties

The heterologous moiety or moieties of the chimeric molecules disclosedherein can comprise, consist of, or consist essentially of prophylacticand/or therapeutic agents (e.g., clotting factors), molecules capable ofimproving a pharmacokinetic (PK) property (e.g., plasma half-lifeextending moieties), detectable moieties (e.g., fluorescent molecules orradionuclides), etc.

As used herein, the term “therapeutic agent” refers to any biological orchemical agent used in the treatment of a disease or disorder.Therapeutic agents include any suitable biologically active chemicalcompounds, biologically derived components such as cells, peptides,antibodies, and polynucleotides, and radiochemical therapeutic agentssuch as radioisotopes. In some embodiments, the chimeric moleculecomprises a clotting factor.

In some embodiments, a heterologous moiety can modify a physicochemicalproperty of a chimeric molecule lacking such heterologous moiety, forexample, it can increase the hydrodynamic radius of a chimeric molecule.In other embodiments, the incorporation of a heterologous moiety into achimeric molecule can improve one or more pharmacokinetic propertieswithout significantly affecting its biological activity or function(e.g., procoagulant activity in chimeric molecules comprising a clottingfactor).

In some embodiments, the heterologous moiety is a polypeptidecomprising, consisting essentially of, or consisting of at least about10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 4000 aminoacids. In other embodiments, the heterologous moiety is a polypeptidecomprising, consisting essentially of, or consisting of about 100 toabout 200 amino acids, about 200 to about 300 amino acids, about 300 toabout 400 amino acids, about 400 to about 500 amino acids, about 500 toabout 600 amino acids, about 600 to about 700 amino acids, about 700 toabout 800 amino acids, about 800 to about 900 amino acids, or about 900to about 1000 amino acids.

In other embodiments, a heterologous moiety increases stability of thechimeric molecule of the invention or a fragment thereof. As usedherein, the term “stability” refers to an art-recognized measure of themaintenance of one or more physical properties of the chimeric moleculein response to an environmental condition (e.g., an elevated or loweredtemperature). In certain embodiments, the physical property can be themaintenance of the covalent structure of the chimeric molecule (e.g.,the absence of proteolytic cleavage, unwanted oxidation or deamidation).In other embodiments, the physical property can also be the presence ofthe chimeric molecule in a properly folded state (e.g., the absence ofsoluble or insoluble aggregates or precipitates). In one embodiment, thestability of the chimeric molecule is measured by assaying a biophysicalproperty of the chimeric molecule, for example thermal stability, pHunfolding profile, stable removal of glycosylation, solubility,biochemical function (e.g., ability to bind to a protein, receptor orligand), etc., and/or combinations thereof. In another embodiment,biochemical function is demonstrated by the binding affinity of theinteraction. In one embodiment, a measure of protein stability isthermal stability, i.e., resistance to thermal challenge. Stability canbe measured using methods known in the art, such as, HPLC (highperformance liquid chromatography), SEC (size exclusion chromatography),DLS (dynamic light scattering), etc. Methods to measure thermalstability include, but are not limited to differential scanningcalorimetry (DSC), differential scanning fluorimetry (DSF), circulardichroism (CD), and thermal challenge assay.

III.A.1 Clotting Factors

In some embodiments, chimeric molecules of the invention comprises atleast one polypeptide heterologous moiety which is (i) a clottingfactor, or (ii) a procoagulant peptide (e.g., a synthetic procoagulantpeptide). In some embodiments, the clotting factor is independentlyselected from the group consisting of factor FVII (“FVII”), factor IX(“FIX”), or factor X (“FX”), and any combinations thereof. As discussedin detail below, the clotting factor can be, for example, FVII zymogen,activatable FVII, activated FVII (FVIIa), FIX zymogen, activatable FIX,activated FIX (FIXa), FX zymogen, activatable FX, or activated FX (FXa).In some embodiments, the clotting factor can comprise a singlepolypeptide chain or two polypeptide chains (e.g., the heavy chain andthe light chain of FVII). The term “activatable clotting factor” refersto a clotting factor in an inactive form (e.g., in its zymogen form)that is capable of being converted to an active form.

As used herein, the term “clotting factor” refers to molecules, oranalogs thereof, naturally occurring or recombinantly produced whichprevent or decrease the duration of a bleeding episode in a subject. Inother words, it means molecules having pro-clotting activity, i.e., areresponsible for the conversion of fibrinogen into a mesh of insolublefibrin causing the blood to coagulate or clot. The term “clottingfactor,” as used herein encompasses clotting factors (e.g., vWF, FV,FVa, FVII, FVIIa, FVIII, FVIIIa, FIX, FIXa, FX, FXa, FXI, FXIa, FXII,FXIIa, FXIII, or FXIIIa), fragments, variants, analogs, or derivativesthereof, naturally occurring, recombinantly produced, or syntheticallyproduced which prevent or decrease the duration of a bleeding episode ina subject. In some embodiments, the chimeric molecule comprises a FVIIor activated FVII (FVIIa) clotting factor. In some embodiments, thechimeric molecule of the invention comprises a FIX or activated FIX(FIXa) clotting factor. In other embodiments, the chimeric moleculecomprises a FX or activated FX (FXa) clotting factor.

In some embodiments, the chimeric molecule comprises a single clottingfactor, which in the chimeric molecule is represented by a formula as H,H1 or H2. In other embodiments, the chimeric molecule comprises twoclotting factors. In some embodiments, the two clotting factors are thesame, whereas in other embodiments, the two clotting factors aredifferent. In some embodiments, one clotting factor is a fragment of aclotting factor (e.g., a heavy chain of a clotting factor such as FVII)and the second clotting factor is a fragment of the same clotting factor(e.g., a light chain of a clotting factor such as FVIII). In someembodiments, the chimeric molecule comprises more than two clottingfactors.

III.A.1.a Factor VII

In some embodiments, the chimeric molecule comprises a clotting factorwhich is a mature form of Factor VII or a variant thereof. Factor VII(FVII, F7; also referred to as Factor 7, coagulation factor VII, serumfactor VII, serum prothrombin conversion accelerator, SPCA, proconvertinand eptacog alpha) is a serine protease that is part of the coagulationcascade. FVII includes a Gla domain, two EGF domains (EGF-1 and EGF-2),and a serine protease domain (or peptidase S1 domain) that is highlyconserved among all members of the peptidase S1 family of serineproteases, such as for example with chymotrypsin. FVII occurs as asingle chain zymogen, an activated zymogen-like two-chain polypeptideand a fully activated two-chain form.

As used herein, a “zymogen-like” protein or polypeptide refers to aprotein that has been activated by proteolytic cleavage, but stillexhibits properties that are associated with a zymogen, such as, forexample, low or no activity, or a conformation that resembles theconformation of the zymogen form of the protein. For example, when it isnot bound to tissue factor, the two-chain activated form of FVII is azymogen-like protein; it retains a conformation similar to the uncleavedFVII zymogen, and, thus, exhibits very low activity. Upon binding totissue factor, the two-chain activated form of FVII undergoesconformational change and acquires its full activity as a coagulationfactor.

Exemplary FVII variants include those with increased specific activity,e.g., mutations that increase the activity of FVII by increasing itsenzymatic activity (Kcat or Km). Such variants have been described inthe art and include, e.g., mutant forms of the molecule as described forexample in Persson et al., Proc. Natl. Acad Sci. USA 98:13583 (2001);Petrovan and Ruf, J Biol. Chem. 276:6616 (2001); Persson et al., J Biol.Chem. 276:29195 (2001); Soejima et al., J Biol. Chem. 276:17229 (2001);Soejima et al., J Biol. Chem. 247:49027 (2002).

In one embodiment, a variant form of FVII includes mutations, e.g.,V158D-E296V-M298Q. In another embodiment, a variant form of FVIIincludes a replacement of amino acids 608-619 (LQQSRKVGDSPN,corresponding to the 170-loop) from the FVII mature sequence with aminoacids EASYPGK (SEQ ID NO: 188) from the 170-loop of trypsin. Highspecific activity variants of FVII are also known in the art. Forexample, Simioni et al. (N.E. Journal of Medicine 361:1671, 2009)describe an R338L mutation. Chang et al. (J. Biol. Chem. 273:12089,1988) and Pierri et al. (Human Gene Therapy 20:479, 2009) describe anR338A mutation. Other mutations are known in the art and include thosedescribed, e.g., in Zogg and Brandstetter, Structure 17:1669 (2009);Sichler et al., J Biol. Chem. 278:4121 (2003); and Sturzebecher et al.,FEBSLett. 412:295 (1997). The contents of these references areincorporated herein by reference.

Full activation, which occurs upon conformational change from azymogen-like form, occurs upon binding to its co-factor, i.e., tissuefactor. Also, mutations can be introduced that result in theconformation change in the absence of tissue factor. Hence, reference toFVIIa includes both two-chain forms thereof: the zymogen-like form, andthe fully activated two-chain form.

III.A.1.b Factor IX

In one embodiment, the chimeric molecule comprises a clotting factorwhich is a mature form of Factor IX or a variant thereof. Factor IXcirculates as a 415 amino acid, single chain plasma zymogen. See,Vysotchin et al., J. Biol. Chem. 268:8436 (1993). The zymogen of FIX isactivated by FXIa or by the tissue factor/FVIIa complex. Specificcleavages between arginine-alanine 145-146 and arginine-valine 180-181result in a light chain and a heavy chain linked by a single disulfidebond between cysteine 132 and cysteine 289 (Bajaj et al., Biochemistry22:4047 (1983)).

The structural organization of FIX is similar to that of the vitaminK-dependent blood clotting proteins FVII, FX and protein C. Theapproximately 45 amino acids of the amino terminus comprise thegamma-carboxyglutamic acid, or Gla, domain. This is followed by twoepidermal growth factor homology domains (EGF), an activation peptideand the catalytic “heavy chain” which is a member of the serine proteasefamily (Vysotchin et al., J Biol. Chem. 268:8436 (1993); Spitzer et al.,Biochemical Journal 265:219 (1990); Brandstetter et al., Proc. Natl.Acad Sci. USA 92:9796 (1995)).

III.A.1.c Factor X

In one embodiment, the chimeric molecule comprises a clotting factorwhich is a mature form of Factor X. Factor X is a vitamin-K dependentglycoprotein with a molecular weight of 58.5 kDa, which is secreted fromliver cells into the plasma as a zymogen. Initially factor X is producedas a prepropeptide with a signal peptide consisting in total of 488amino acids. The signal peptide is cleaved off by signal peptidaseduring export into the endoplasmatic reticulum. The propeptide sequenceis cleaved off after gamma carboxylation took place at the first 11glutamic acid residues at the N-terminus of the mature N-terminal chain.A further processing step occurs by cleavage between Arg182 and Ser183.This processing step also leads concomitantly to the deletion of thetripeptide Arg180-Lys181-Arg182. The resulting secreted factor X zymogenconsists of an N-terminal light chain of 139 amino acids (M, 16,200) anda C-terminal heavy chain of 306 amino acids (M, 42,000) which arecovalently linked via a disulfide bridge between Cys172 and Cys342.Further posttranslational processing steps include the 3-hydroxylationof Asp103 as well as N- and O-type glycosylation.

It will be understood that in addition to wild type (WT) versions ofthese clotting factors or biologically active portions thereof, theheterologous moieties in the chimeric molecules disclosed herein canalso comprise precursor truncated forms thereof that have activity,allelic variants and species variants, variants encoded by splicevariants, and other variants, including polypeptides that have at least40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more sequence identity to the mature form of the clotting factorand which retain the ability to promote clot formation. For example,modified FVII polypeptides and variants thereof which retain at leastone activity of FVII, such as TF binding, factor X binding, phospholipidbinding, and/or coagulant activity of FVII can be employed. By retainingactivity, the activity can be altered, such as reduced or increased, ascompared to a wild-type clotting factor so long as the level of activityretained is sufficient to yield a detectable effect.

Exemplary modified polypeptides include, but are not limited to,tissue-specific isoforms and allelic variants thereof, syntheticmolecules prepared by translation of nucleic acids, proteins generatedby chemical synthesis, such as syntheses that include ligation ofshorter polypeptides, through recombinant methods, proteins isolatedfrom human and non-human tissue and cells, chimeric polypeptides andmodified forms thereof. The instant clotting factors can also consist offragments or portions of WT molecules that are of sufficient length orinclude appropriate regions to retain at least one activity (uponactivation if needed) of a full-length mature polypeptide. Exemplaryclotting factor variants are known in the art.

As used herein, the term “Gla domain” refers to the conserved membranebinding motif which is present in vitamin K-dependent proteins, such asprothrombin, coagulation factors VII, IX and X, proteins C, S, and Z.These proteins require vitamin K for the posttranslational synthesis ofγ-carboxyglutamic acid, an amino acid clustered in the N-terminal Gladomain of these proteins. All glutamic residues present in the domainare potential carboxylation sites and many of them are thereforemodified by carboxylation. In the presence of calcium ions, the Gladomain interacts with phospholipid membranes that includephosphatidylserine. The Gla domain also plays a role in binding to theFVIIa cofactor, tissue factor (TF). Complexed with TF, the Gla domain ofFVIIa is loaded with seven Ca²⁺ ions, projects three hydrophobic sidechains in the direction of the cell membrane for interaction withphospholipids on the cell surface, and has significant contact with theC-terminal domain of TF.

The Gla domain of factor VII comprises the uncommon amino acidγ-carboxyglutamic acid (Gla), which plays a vital role in the binding ofclotting factors to negatively charged phospholipid surfaces. The Gladomain is responsible for the high-affinity binding of calcium ions. Itstarts at the N-terminal extremity of the mature form of proteins andends with a conserved aromatic residue. A conserved Gla-x(3)-Gla-x-Cysmotif is found in the middle of the domain which seems to be importantfor substrate recognition by the carboxylase. Using stopped-flowfluorescence kinetic measurements in combination with surface plasmonresonance analysis, the Gla domain has been found to be important in thesequence of events whereby the protease domain of FVIIa initiatescontact with sTF (Osterlund et al., Biochem. Biophys. Res. Commun.337:1276 (2005)). In addition, clearance of clotting factors can besignificantly mediated through Gla interactions, e.g., on liver cellsand clearance receptors, e.g., EPCR.

In one embodiment, the chimeric molecule comprises a heterologous moietycomprising a clotting factor modified to lack a Gla domain. The Gladomain is responsible for mediating clearance of clotting factors viamultiple pathways, such as binding to liver cells, clearance receptorssuch as EPCR, etc. Thus, eliminating the Gla domain has beneficialeffects on half life of clotting factors. Though Gla domain is alsogenerally required for activity by localizing clotting factors to sitesof coagulation, the inclusion of a platelet targeting domain moiety(e.g., a GPIIb/IIIa antibody or antigen-binding molecule thereof)targets the Gla deleted clotting factor to platelets. Accordingly, inone embodiment, the chimeric molecule comprises a targeting moiety(e.g., a GPIIb/IIIa antibody or antigen-binding molecule thereof) and aheterologous moiety comprising a clotting factor that lacks a Gladomain. For example, in the case of Factor VII, the Gla domain ispresent at the amino terminus of the light chain and consists of aminoacids 1-35. The Gla domains of the exemplary clotting factors disclosedherein are known in the art. The Gla domain can be removed usingstandard molecular biology techniques, replaced with a targeting domain,and the modified light chain incorporated into a construct of theinvention. In one embodiment, a cleavage site can be introduced intoconstructs lacking a Gla domain to facilitate activation of themolecule. For example, in one embodiment, such a cleavage site can beintroduced between the amino acids that are cleaved when the clottingfactor is activated (e.g., between amino acids 152 and 153 in the caseof Factor VII).

In one embodiment, a cleavage site can be introduced into chimericmolecules comprising a clotting factor that lacks a Gla domain tofacilitate activation of the molecule. For example, in one embodiment,such a cleavage site can be introduced between the amino acids that arecleaved when the clotting factor is activated (e.g., between amino acids152 and 153 in the case of Factor VII). Exemplary clotting factorslacking a Gla domain are known in the art. Exemplary clotting factorsare those of mammalian, e.g., human, origin.

III.A.2 Half-Life Extending Moieties

In some embodiments, the chimeric molecule comprises at last oneheterologous moiety that is a “half-life extending moiety.” As usedherein, the term “half-life extending moiety” refers to a heterologousmoiety which increases the in vivo half-life of a protein, for example,a chimeric molecule. The term “half-life” refers to a biologicalhalf-life of a particular protein or polypeptide (e.g., a clottingfactor or a chimeric molecule disclosed herein) in vivo. Half-life canbe represented by the time required for half the quantity administeredto a subject to be cleared from the circulation and/or other tissues inthe animal. When a clearance curve of a given polypeptide or chimericmolecule of the invention is constructed as a function of time, thecurve is usually biphasic with a rapid α-phase and longer β-phase. Theα-phase typically represents an equilibration of the administered Fcpolypeptide between the intra- and extra-vascular space and is, in part,determined by the size of the polypeptide. The β-phase typicallyrepresents the catabolism of the polypeptide in the intravascular space.In some embodiments, procoagulant compounds of the invention aremonophasic, and thus do not have an alpha phase, but just the singlebeta phase. In certain embodiments, the term half-life as used hereinrefers to the half-life of the procoagulant compound in the β-phase. Thetypical 3 phase half-life of a human antibody in humans is 21 days. Invivo half-life of a chimeric molecule can be determined by any methodknown to those of skill in the art. In certain embodiments, thehalf-life extending moiety can comprise an attachment site for anon-polypeptide moiety (e.g., PEG).

Half-life extending moieties, as discussed below in detail, cancomprise, for example, (i) low complexity peptides, (ii) albumin, (iii)albumin binding polypeptide or fatty acid, (iv) Fc, (v) transferrin,(vi) PAS, (vii) the C-terminal peptide (CTP) of the β subunit of humanchorionic gonadotropin, (viii) polyethylene glycol (PEG), (ix)hydroxyethyl starch (HES), (x) albumin-binding small molecules, (xi)vWF, (xii) a clearance receptor or fragment thereof which blocks bindingof the chimeric molecule to a clearance receptor, or (xiii) anycombinations thereof. In some embodiments, the half-life extendingmoiety comprises an Fc region. In other embodiments, the half-lifeextending moiety comprises two Fc regions fused by a linker. Exemplaryheterologous moieties also include, e.g., FcRn binding moieties (e.g.,complete Fc regions or portions thereof which bind to FcRn), singlechain Fc regions (scFc regions, e.g., as described in U.S. Publ. No.2008-0260738, and Intl. Publ. Nos. WO 2008-012543 and WO 2008-1439545),or processable scFc regions. In some embodiments, a heterologous moietycan include an attachment site for a non-polypeptide moiety such aspolyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid,or any derivatives, variants, or combinations of these moieties.

In certain embodiments, a chimeric molecule of the invention comprisesat least one half-like extending moiety which increases the in vivohalf-life of the chimeric molecule with respect to the in vivo half-lifeof the corresponding chimeric molecule lacking such heterologous moiety.In vivo half-life of a chimeric molecule can be determined by any methodknown to those of skill in the art, e.g., activity assays (chromogenicassay or one stage clotting aPTT assay), ELISA, etc.

In some embodiments, the presence of one or more half-life extendingmoiety results in the half-life of the chimeric molecule to be increasedcompared to the half-life of the corresponding chimeric molecule lackingsuch one or more half-life extending moieties. The half-life of thechimeric molecule comprising a half-life extending moiety is at leastabout 1.5 times, at least about 2 times, at least about 2.5 times, atleast about 3 times, at least about 4 times, at least about 5 times, atleast about 6 times, at least about 7 times, at least about 8 times, atleast about 9 times, at least about 10 times, at least about 11 times,or at least about 12 times longer than the in vivo half-life of thecorresponding chimeric molecule lacking such half-life extending moiety.

In one embodiment, the half-life of the chimeric molecule comprising ahalf-life extending moiety is about 1.5-fold to about 20-fold, about 1.5fold to about 15 fold, or about 1.5 fold to about 10 fold longer thanthe in vivo half-life of the corresponding chimeric molecule lackingsuch half-life extending moiety. In another embodiment, the half-life ofchimeric molecule comprising a half-life extending moiety is extendedabout 2-fold to about 10-fold, about 2-fold to about 9-fold, about2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-fold toabout 6-fold, about 2-fold to about 5-fold, about 2-fold to about4-fold, about 2-fold to about 3-fold, about 2.5-fold to about 10-fold,about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about 2.5-foldto about 5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about3-fold, about 3-fold to about 10-fold, about 3-fold to about 9-fold,about 3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-foldto about 6-fold, about 3-fold to about 5-fold, about 3-fold to about4-fold, about 4-fold to about 6 fold, about 5-fold to about 7-fold, orabout 6-fold to about 8 fold as compared to the in vivo half-life of thecorresponding chimeric molecule lacking such half-life extending moiety.

III.A.2.a Fc Region

In certain embodiments, the chimeric molecule comprises at least aheterologous moiety comprising a Fc region. “Fc” or “Fc region” as usedherein means a functional neonatal Fc receptor (FcRn) binding partnercomprising an Fc domain, variant, or fragment thereof, unless otherwisespecified. An FcRn binding partner is any molecule that can bespecifically bound by the FcRn receptor with consequent active transportby the FcRn receptor of the FcRn binding partner. Thus, the term Fcincludes any variants of IgG Fc that are functional. The region of theFc portion of IgG that binds to the FcRn receptor has been describedbased on X-ray crystallography (Burmeister et al., Nature 372:379(1994), incorporated herein by reference in its entirety). The majorcontact area of the Fc with the FcRn is near the junction of the CH2 andCH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain.FcRn binding partners include, but are not limited to, whole IgG, the Fcfragment of IgG, and other fragments of IgG that include the completebinding region of FcRn. An Fc can comprise the CH2 and CH3 domains of animmunoglobulin with or without the hinge region of the immunoglobulin.Also included are Fc fragments, variants, or derivatives which maintainthe desirable properties of an Fc region in a chimeric molecule, e.g.,an increase in half-life, e.g., in vivo half-life. Myriad mutants,fragments, variants, and derivatives are described, e.g., in PCTPublication Nos. WO2011/069164, WO2012/006623, WO2012/006635, or WO2012/006633, all of which are incorporated herein by reference in theirentireties.

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and an Fc region.

III.A.2.b scFc (Single Chain Fc) Region

In one embodiment, the chimeric molecule comprises a heterologous moietycomprising one genetically fused Fc region or a portion thereof within asingle polypeptide chain (i.e., a single-chain Fc (scFc) region). Theunprocessed polypeptides comprise at least two immunoglobulin constantregions or portions thereof (e.g., Fc moieties or domains (e.g., 2, 3,4, 5, 6, or more Fc moieties or domains)) within the same linearpolypeptide chain that are capable of folding (e.g., intramolecularly orintermolecularly folding) to form one functional scFc region which islinked by an Fc peptide linker. For example, in one embodiment, apolypeptide of the invention is capable of binding, via its scFc region,to at least one Fc receptor (e.g., an FcRn, an FcγR receptor (e.g.,FcγRIII), or a complement protein (e.g., C1q)) in order to improvehalf-life or trigger an immune effector function (e.g.,antibody-dependent cytotoxicity (ADCC), phagocytosis, orcomplement-dependent cytotoxicity (CDCC) and/or to improvemanufacturability).

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and an scFc region.

III.A.2.c Albumins

In certain embodiments, the chimeric molecule comprises a heterologousmoiety comprising albumin or a functional fragment thereof. Human serumalbumin (HSA, or HA), a protein of 609 amino acids in its full-lengthform, is responsible for a significant proportion of the osmoticpressure of serum and also functions as a carrier of endogenous andexogenous ligands. The term “albumin” as used herein includesfull-length albumin or a functional fragment, variant, derivative, oranalog thereof. Examples of albumin or the fragments or variants thereofare disclosed in US Pat. Publ. Nos. US2008/0194481, US2008/0004206,US2008/0161243, US2008/0261877, or US2008/0153751 or PCT Appl. Publ.Nos. WO2008/033413, WO2009/058322, or WO2007/021494, which areincorporated herein by reference in their entireties.

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and an albumin.

III.A.2.d Albumin Binding Polypeptides and Lipids

In certain embodiments, a heterologous moiety can comprise an albuminbinding moiety, which comprises an albumin binding peptide, a bacterialalbumin binding domain, an albumin-binding antibody fragment, or anycombinations thereof. For example, the albumin binding protein can be abacterial albumin binding protein, an antibody or an antibody fragmentincluding domain antibodies (see, e.g., U.S. Pat. No. 6,696,245). Analbumin binding protein, for example, can be a bacterial albumin bindingdomain, such as the one of streptococcal protein G (Konig and Skerra(1998) J Immunol. Methods 218, 73-83). Other examples of albumin bindingpeptides that can be used as conjugation partner are, for instance,those having a Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, whereinXaa₁ is Asp, Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, H is, Ile, Leu, orLys; Xaa₃ is Ala, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe,Ser, or Thr as described in U.S. Pub. No. US2003/0069395 or Dennis etal. (2002) J. Biol. Chem. 277, 35035-35043.

Domain 3 from streptococcal protein G, as disclosed by Kraulis et al.,FEBS Lett. 378:190-194 (1996) and Linhult et al., Protein Sci.11:206-213 (2002) is an example of a bacterial albumin-binding domain.Examples of albumin-binding peptides include a series of peptides havingthe core sequence DICLPRWGCLW (SEQ ID NO: 162). See, e.g., Dennis etal., J. Biol. Chem. 2002, 277: 35035-35043 (2002). Examples ofalbumin-binding antibody fragments are disclosed in Muller andKontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007); Roovers et al.,Cancer Immunol. Immunother. 56:303-317 (2007), and Holt et al., Prot.Eng. Design Sci., 21:283-288 (2008), which are incorporated herein byreference in their entireties. An example of such albumin binding moietyis 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido) hexanoate(“Albu” tag) as disclosed by Trussel et al., Bioconjugate Chem.20:2286-2292 (2009). Fatty acids, in particular long chain fatty acids(LCFA) and long chain fatty acid-like albumin-binding compounds can beused to extend the in vivo half-life of chimeric molecules of theinvention. An example of a LCFA-like albumin-binding compound is16-(1-(3-(9-(((2,5-dioxopyrrolidin-1-yloxy)carbonyloxy)-methyi)-7-sulfo-9H-fluoren-2-ylamino)-3-oxopropyl)-2,5-dioxopyrrolidin-3-ylthio)hexadecanoic acid (see, e.g., WO 2010/140148).

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and an albumin binding polypeptide orlipid.

III.A.2.e CTP

In certain embodiments, a chimeric molecule disclosed herein comprisesat least one heterologous moiety comprising one β subunit of theC-terminal peptide (CTP) of human chorionic gonadotropin or fragment,variant, or derivative thereof. The insertion of one or more CTPpeptides into a recombinant protein is known to increase the in vivohalf-life of that protein. See, e.g., U.S. Pat. No. 5,712,122,incorporated by reference herein in its entirety.

Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ IDNO: 153) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 154). See, e.g.,U.S. Patent Appl. Publ. No. US 2009/0087411, incorporated by reference.In some embodiments, the chimeric molecule comprises two heterologousmoieties that are CTP sequences. In some embodiments, three of theheterologous moieties are CTP sequences. In some embodiments, four ofthe heterologous moieties are CTP sequences. In some embodiments, fiveof the heterologous moieties are CTP sequences. In some embodiments, sixor more of the heterologous moieties are CTP sequences.

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and a CTP.

III.A.2.f PAS

In other embodiments, at least one heterologous moiety is a PASsequence. A PAS sequence, as used herein, means an amino acid sequencecomprising mainly alanine and serine residues or comprising mainlyalanine, serine, and proline residues, the amino acid sequence formingrandom coil conformation under physiological conditions. Accordingly,the PAS sequence is a building block, an amino acid polymer, or asequence cassette comprising, consisting essentially of, or consistingof alanine, serine, and proline which can be used as a part of theheterologous moiety in the chimeric molecule. Yet, the skilled person isaware that an amino acid polymer also can form random coil conformationwhen residues other than alanine, serine, and proline are added as aminor constituent in the PAS sequence.

The term “minor constituent” as used herein means that amino acids otherthan alanine, serine, and proline can be added in the PAS sequence to acertain degree, e.g., up to about 12%, i.e., about 12 of 100 amino acidsof the PAS sequence, up to about 10%, i.e., about 10 of 100 amino acidsof the PAS sequence, up to about 9%, i.e., about 9 of 100 amino acids,up to about 8%, i.e., about 8 of 100 amino acids, about 6%, i.e., about6 of 100 amino acids, about 5%, i.e., about 5 of 100 amino acids, about4%, i.e., about 4 of 100 amino acids, about 3%, i.e., about 3 of 100amino acids, about 2%, i.e., about 2 of 100 amino acids, about 1%, i.e.,about 1 of 100 of the amino acids.

The amino acids different from alanine, serine and proline can beselected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,Met, Phe, Thr, Trp, Tyr, or Val.

Under physiological conditions, the PAS sequence stretch forms a randomcoil conformation and thereby can mediate an increased in vivo and/or invitro stability to the chimeric molecule. Since the random coil domaindoes not adopt a stable structure or function by itself, the biologicalactivity mediated by the activatable clotting factor in the chimericmolecule is essentially preserved. In other embodiments, the PASsequences that form random coil domain are biologically inert,especially with respect to proteolysis in blood plasma, immunogenicity,isoelectric point/electrostatic behavior, binding to cell surfacereceptors or internalization, but are still biodegradable, whichprovides clear advantages over synthetic polymers such as PEG.

Non-limiting examples of the PAS sequences forming random coilconformation comprise an amino acid sequence selected from the groupconsisting of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 155),AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 156), APSSPSPSAPSSPSPASPSS (SEQ ID NO:157), APSSPSPSAPSSPSPASPS (SEQ ID NO: 158), SSPSAPSPSSPASPSPSSPA (SEQ IDNO: 159), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 160) andASAAAPAAASAAASAPSAAA (SEQ ID NO: 161) or any combinations thereof.Additional examples of PAS sequences are known from, e.g., US Pat. Publ.No. 2010/0292130 and PCT Appl. Publ. No. WO2008/155134 A1.

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and a PAS.

III.A.2.g HAP

In certain embodiments, at least one heterologous moiety is aglycine-rich homo-amino-acid polymer (HAP). The HAP sequence cancomprise a repetitive sequence of glycine, which has at least 50 aminoacids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500amino acids in length. In one embodiment, the HAP sequence is capable ofextending half-life of a moiety fused to or linked to the HAP sequence.Non-limiting examples of the HAP sequence includes, but are not limitedto (Gly)_(n), (Gly4Ser)_(n) or S(Gly4Ser)_(n), wherein n is 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In oneembodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. See,e.g., Schlapschy M et al., Protein Eng. Design Selection, 20: 273-284(2007).

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and a HAP.

III.A.2.h Transferrin

In certain embodiments, at least one heterologous moiety is transferrinor a peptide or fragment, variant, or derivative thereof. Anytransferrin can be used to make the chimeric molecules of the invention.As an example, wild-type human TF (TF) is a 679 amino acid protein, ofapproximately 75 KDa (not accounting for glycosylation), with two maindomains, N (about 330 amino acids) and C (about 340 amino acids), whichappear to originate from a gene duplication. N domain comprises twosubdomains, N1 domain and N2 domain, and C domain comprises twosubdomains, C1 domain and C2 domain. See GenBank accession numbersNM001063, XM002793, M12530, XM039845, XM 039847 and S95936(www.ncbi.nlm.nih.gov), all of which are herein incorporated byreference in their entirety. In one embodiment, the transferrinheterologous moiety includes a transferrin splice variant. In oneexample, a transferrin splice variant can be a splice variant of humantransferrin, e.g., Genbank Accession AAA61140. In another embodiment,the transferrin portion of the chimeric molecule includes one or moredomains of the transferrin sequence, e.g., N domain, C domain, N1domain, N2 domain, C1 domain, C2 domain or any combinations thereof.

Transferrin transports iron through transferrin receptor (TfR)-mediatedendocytosis. After the iron is released into an endosomal compartmentand Tf-TfR complex is recycled to cell surface, the Tf is released backextracellular space for next cycle of iron transporting. Tf possesses along half-life that is in excess of 14-17 days (Li et al., TrendsPharmacol. Sci. 23:206-209 (2002)). Transferrin fusion proteins havebeen studied for half-life extension, targeted deliver for cancertherapies, oral delivery and sustained activation of proinsulin(Brandsma et al., Biotechnol. Adv., 29: 230-238 (2011); Bai et al.,Proc. Natl. Acad. Sci. USA 102:7292-7296 (2005); Kim et al., J.Pharmacol. Exp. Ther., 334:682-692 (2010); Wang et al., J. ControlledRelease 155:386-392 (2011)).

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and a transferrin.

III.A.2.i PEG

In some embodiments, at least one heterologous moiety is a solublepolymer known in the art, including, but not limited to, polyethyleneglycol, ethylene glycol/propylene glycol copolymers,carboxymethylcellulose, dextran, or polyvinyl alcohol. In someembodiments, the chimeric molecule comprising a PEG heterologous moietyfurther comprises a heterologous moiety selected from an immunoglobulinconstant region or portion thereof (e.g., an Fc region), a PAS sequence,HES, and albumin, fragment, or variant thereof. In still otherembodiments, the chimeric molecule comprises an activatable clottingfactor or fragment thereof and a PEG heterologous moiety, wherein thechimeric molecule further comprises a heterologous moiety selected froman immunoglobulin constant region or portion thereof (e.g., an Fcmoiety), a PAS sequence, HES, and albumin, fragment, or variant thereof.In yet other embodiments, the chimeric molecule comprises a clottingfactor or fragment thereof, a second clotting factor or fragmentthereof, and a PEG heterologous moiety, wherein the chimeric moleculefurther comprises a heterologous moiety selected from an immunoglobulinconstant region or portion thereof (e.g., an Fc moiety), a PAS sequence,HES, and albumin, fragment, or variant thereof.

In other embodiments, the chimeric molecule comprises a clotting factoror fragment thereof, a synthetic procoagulant polypeptide, and a PEGheterologous moiety, wherein the chimeric molecule further comprises aheterologous moiety selected from an immunoglobulin constant region orportion thereof (e.g., an Fc region), a PAS sequence, HES, and albumin,fragment, or variant thereof. In other embodiments, the chimericmolecule comprises two synthetic procoagulant peptides and a PEGheterologous moiety, wherein the chimeric molecule further comprises aheterologous moiety selected from the group consisting of animmunoglobulin constant region or portion thereof (e.g., an Fc region),a PAS sequence, HES, and albumin, fragment, or variant thereof. In yetanother embodiment, the chimeric molecule comprises a clotting factor orfragment thereof, a clotting factor cofactor (e.g., Tissue Factor if theclotting factor is Factor VII), and a PEG heterologous moiety, whereinthe chimeric molecule further comprises a heterologous moiety selectedfrom an immunoglobulin constant region or portion thereof (e.g., an Fcregion), a PAS sequence, HES, and albumin, fragment, or variant thereof.

The polymer can be of any molecular weight, and can be branched orunbranched. For polyethylene glycol, in one embodiment, the molecularweight is between about 1 kDa and about 100 kDa for ease in handling andmanufacturing. Other sizes can be used, depending on the desired profile(e.g., the duration of sustained release desired, the effects, if any onbiological activity, the ease in handling, the degree or lack ofantigenicity and other known effects of the polyethylene glycol to aprotein or analog). For example, the polyethylene glycol can have anaverage molecular weight of about 200, 500, 1000, 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000,13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500,18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,90,000, 95,000, or 100,000 kDa.

In some embodiments, the polyethylene glycol can have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), each ofwhich is incorporated herein by reference in its entirety.

The number of polyethylene glycol moieties attached to each chimericmolecule of the invention (i.e., the degree of substitution) can alsovary. For example, the PEGylated chimeric molecule can be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

In some embodiments, the chimeric molecule can be PEGylated. A PEGylatedchimeric molecule comprises at least one polyethylene glycol (PEG)molecule. In other embodiments, the polymer can be water-soluble.Non-limiting examples of the polymer can be poly(alkylene oxide),poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, orpoly(acryloylmorpholine). Additional types of polymer-conjugation toclotting factors are disclosed in U.S. Pat. No. 7,199,223. See also,Singh et al. Curr. Med. Chem. 15:1802-1826 (2008).

There are a number of PEG attachment methods available to those skilledin the art, for example Malik F et al., Exp. Hematol. 20:1028-35 (1992);Francis, Focus on Growth Factors 3(2):4-10 (1992); European Pat. Pub.Nos. EP0401384, EP0154316, and EP0401384; and International Pat. Appl.Pub. Nos. WO92/16221 and WO95/34326.

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and a PEG.

III.A.2.j HES

In certain embodiments, at least one heterologous moiety is a polymer,e.g., hydroxyethyl starch (HES) or a derivative thereof. Hydroxyethylstarch (HES) is a derivative of naturally occurring amylopectin and isdegraded by alpha-amylase in the body. HES is a substituted derivativeof the carbohydrate polymer amylopectin, which is present in corn starchat a concentration of up to 95% by weight. HES exhibits advantageousbiological properties and is used as a blood volume replacement agentand in hemodilution therapy in the clinics (Sommermeyer et al.,Krankenhauspharmazie, 8(8), 271-278 (1987); and Weidler et al.,Arzneim.-Forschung/Drug Res., 41, 494-498 (1991)).

Amylopectin contains glucose moieties, wherein in the main chainalpha-1,4-glycosidic bonds are present and at the branching sitesalpha-1,6-glycosidic bonds are found. The physical-chemical propertiesof this molecule are mainly determined by the type of glycosidic bonds.Due to the nicked alpha-1,4-glycosidic bond, helical structures withabout six glucose-monomers per turn are produced. The physico-chemicalas well as the biochemical properties of the polymer can be modified viasubstitution. The introduction of a hydroxyethyl group can be achievedvia alkaline hydroxyethylation. By adapting the reaction conditions itis possible to exploit the different reactivity of the respectivehydroxy group in the unsubstituted glucose monomer with respect to ahydroxyethylation. Owing to this fact, the skilled person is able toinfluence the substitution pattern to a limited extent.

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. The degree of substitution, denoted as DS,relates to the molar substitution, is known to the skilled people. SeeSommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), as citedabove, in particular p. 273.

In one embodiment, hydroxyethyl starch has a mean molecular weight(weight mean) of from 1 to 300 kD, from 2 to 200 kD, from 3 to 100 kD,or from 4 to 70 kD. Hydroxyethyl starch can further exhibit a molardegree of substitution of from 0.1 to 3, preferably 0.1 to 2, morepreferred, 0.1 to 0.9, preferably 0.1 to 0.8, and a ratio between C2:C6substitution in the range of from 2 to 20 with respect to thehydroxyethyl groups. A non-limiting example of HES having a meanmolecular weight of about 130 kD is a HES with a degree of substitutionof 0.2 to 0.8 such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, preferablyof 0.4 to 0.7 such as 0.4, 0.5, 0.6, or 0.7. In a specific embodiment,HES with a mean molecular weight of about 130 kD is VOLUVEN® fromFresenius. VOLUVEN® is an artificial colloid, employed, e.g., for volumereplacement used in the therapeutic indication for therapy andprophylaxis of hypovolemia. The characteristics of VOLUVEN® are a meanmolecular weight of 130,000+/−20,000 D, a molar substitution of 0.4 anda C2:C6 ratio of about 9:1. In other embodiments, ranges of the meanmolecular weight of hydroxyethyl starch are, e.g., 4 to 70 kD or 10 to70 kD or 12 to 70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10to 50 kD or 12 to 50 kD or 18 to 50 kD or 4 to 18 kD or 10 to 18 kD or12 to 18 kD or 4 to 12 kD or 10 to 12 kD or 4 to 10 kD. In still otherembodiments, the mean molecular weight of hydroxyethyl starch employedis in the range of from more than 4 kD and below 70 kD, such as about 10kD, or in the range of from 9 to 10 kD or from 10 to 11 kD or from 9 to11 kD, or about 12 kD, or in the range of from 11 to 12 kD) or from 12to 13 kD or from 11 to 13 kD, or about 18 kD, or in the range of from 17to 18 kD or from 18 to 19 kD or from 17 to 19 kD, or about 30 kD, or inthe range of from 29 to 30, or from 30 to 31 kD, or about 50 kD, or inthe range of from 49 to 50 kD or from 50 to 51 kD or from 49 to 51 kD.

In certain embodiments, the heterologous moiety can be a mixture ofhydroxyethyl starches having different mean molecular weights and/ordifferent degrees of substitution and/or different ratios of C2: C6substitution. Therefore, mixtures of hydroxyethyl starches can beemployed having different mean molecular weights and different degreesof substitution and different ratios of C2: C6 substitution, or havingdifferent mean molecular weights and different degrees of substitutionand the same or about the same ratio of C2:C6 substitution, or havingdifferent mean molecular weights and the same or about the same degreeof substitution and different ratios of C2:C6 substitution, or havingthe same or about the same mean molecular weight and different degreesof substitution and different ratios of C2:C6 substitution, or havingdifferent mean molecular weights and the same or about the same degreeof substitution and the same or about the same ratio of C2:C6substitution, or having the same or about the same mean molecularweights and different degrees of substitution and the same or about thesame ratio of C2:C6 substitution, or having the same or about the samemean molecular weight and the same or about the same degree ofsubstitution and different ratios of C2: C6 substitution, or havingabout the same mean molecular weight and about the same degree ofsubstitution and about the same ratio of C2:C6 substitution.

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and a HES.

III.A.2.k PSA

In certain embodiments, at least one heterologous moiety is a polymer,e.g., polysialic acids (PSAs) or a derivative thereof. Polysialic acids(PSAs) are naturally occurring unbranched polymers of sialic acidproduced by certain bacterial strains and in mammals in certain cellsRoth J., et al. (1993) in Polysialic Acid: From Microbes to Man, edsRoth J., Rutishauser U., Troy F. A. (Birkhauser Verlag, Basel,Switzerland), pp 335-348. They can be produced in various degrees ofpolymerisation from n=about 80 or more sialic acid residues down to n=2by limited acid hydrolysis or by digestion with neuraminidases, or byfractionation of the natural, bacterially derived forms of the polymer.The composition of different polysialic acids also varies such thatthere are homopolymeric forms i.e. the alpha-2,8-linked polysialic acidcomprising the capsular polysaccharide of E. coli strain K1 and thegroup-B meningococci, which is also found on the embryonic form of theneuronal cell adhesion molecule (N-CAM). Heteropolymeric forms alsoexist-such as the alternating alpha-2,8 alpha-2,9 polysialic acid of E.coli strain K92 and group C polysaccharides of N. meningitidis. Sialicacid can also be found in alternating copolymers with monomers otherthan sialic acid such as group W135 or group Y of N. meningitidis.Polysialic acids have important biological functions including theevasion of the immune and complement systems by pathogenic bacteria andthe regulation of glial adhesiveness of immature neurons during foetaldevelopment (wherein the polymer has an anti-adhesive function) Cho andTroy, P.N.A.S., USA, 91 (1994) 11427-11431, although there are no knownreceptors for polysialic acids in mammals. The alpha-2,8-linkedpolysialic acid of E. coli strain K1 is also known as ‘colominic acid’and is used (in various lengths) to exemplify the present invention.Various methods of attaching or conjugating polysialic acids to apolypeptide have been described (for example, see U.S. Pat. No.5,846,951; WO-A-0187922, and US 2007/0191597 A1, which are incorporatedherein by reference in their entireties.

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and a PSA.

III.A.2.1 Clearance Receptors

In certain embodiments, the in vivo half-life of a chimeric molecule ofthe invention can be extended where the chimeric molecule comprises atleast one heterologous molecule comprising a clearance receptor,fragment, variant, or derivative thereof. In specific embodimentswherein the chimeric molecule comprises Factor X, soluble forms ofclearance receptors, such as the low density lipoprotein-related proteinreceptor LRP1, or fragments thereof, can block binding of Factor X toclearance receptors and thereby extend its in vivo half-life.

LRP1 is a 600 kDa integral membrane protein that is implicated in thereceptor-mediate clearance of a variety of proteins, such as FVIII or X.See, e.g., Narita et al., Blood 91:555-560 (1998); Lenting et al.,Haemophilia 16:6-16 (2010). Other suitable clearance receptors are,e.g., LDLR (low-density lipoprotein receptor), VLDLR (very low-densitylipoprotein receptor), and megalin (LRP-2), or fragments thereof. See,e.g., Bovenschen et al., Blood 106:906-912 (2005); Bovenschen, Blood116:5439-5440 (2010); Martinelli et al., Blood 116:5688-5697 (2010).

In some embodiments, the chimeric molecule comprises a clotting factor(e.g., FVII), a targeting moiety (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof), and a clearance receptor, fragment,variant, or derivative thereof.

III.B Linkers

As used herein, the term “linker” or “linker moiety” (represented as L,L1, or L2 in the formulas disclosed herein) refers to a peptide orpolypeptide sequence (e.g., a synthetic peptide or polypeptidesequence), or a non-peptide linker for which its main function is toconnect two domains in a linear amino acid sequence of a polypeptidechain, for example, two heterologous moieties in a chimeric molecule ofthe invention. Accordingly, in some embodiments, linkers are interposedbetween two heterologous moieties, between a heterologous moiety and atargeting moiety, which binds to a platelet (e.g., an anti-GPIIb/IIIaantibody or antigen-binding molecule thereof disclosed herein), betweena clotting factor (either the heavy chain or the light chain) and atargeting moiety, which binds to a platelet (e.g., an anti-GPIIb/IIIaantibody or antigen-binding molecule thereof disclosed herein), orbetween a clotting factor (either the heavy chain or the light chain)and a heterologous moiety.

When multiple linkers are present in a chimeric molecule of theinvention, each of the linkers can be the same or different. Generally,linkers provide flexibility to the chimeric molecule. Linkers are nottypically cleaved; however in certain embodiments, such cleavage can bedesirable. Accordingly, in some embodiments a linker can comprise one ormore protease-cleavable sites, which can be located within the sequenceof the linker or flanking the linker at either end of the sequence ofthe linker.

In some embodiments, the chimeric molecule comprises one or morelinkers, wherein one or more of the linkers comprise a peptide linker.In other embodiments, one or more of the linkers comprise a non-peptidelinker. In some embodiments, the peptide linker can comprise at leasttwo amino, at least three, at least four, at least five, at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, or at least 100 amino acids. In otherembodiments, the peptide linker can comprise at least 200, at least 300,at least 400, at least 500, at least 600, at least 700, at least 800, atleast 900, or at least 1,000 amino acids. In some embodiments, thepeptide linker can comprise at least about 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids.

The peptide linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids,200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 aminoacids, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,1500-1600, 1600-1700, 1700-1800, 1800-1900, or 1900-2000 amino acids.

Examples of peptide linkers are well known in the art, for examplepeptide linkers according to the formula [(Gly)_(x)-Ser_(y)]_(z) where xis from 1 to 4, y is 0 or 1, and z is from 1 to 50. In one embodiment,the peptide linker comprises the sequence G_(n), where n can be aninteger from 1 to 100. In a specific embodiment, the specificembodiment, the sequence of the peptide linker is GGGG. The peptidelinker can comprise the sequence (GA)_(n). The peptide linker cancomprise the sequence (GGS)_(n). In other embodiments, the peptidelinker comprises the sequence (GGGS)_(n) (SEQ ID NO: 166). In stillother embodiments, the peptide linker comprises the sequence(GGS)_(n)(GGGGS)_(n)(SEQ ID NO: 189). In these instances, n can be aninteger from 1-100. In other instances, n can be an integer from 1-20,i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS(SEQ ID NO: 190), GGSGGSGGSGGSGGG (SEQ ID NO: 191), GGSGGSGGGGSGGGGS(SEQ ID NO: 192), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 193), orGGGGSGGGGSGGGGS (SEQ ID NO: 194). In other embodiments, the linker is apoly-G sequence (GGGG)_(n), where n can be an integer from 1-100.

An exemplary Gly/Ser peptide linker comprises the amino acid sequence(Gly4Ser)_(n) (SEQ ID NO: 195), wherein n is an integer that is the sameor higher than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,46, 50, 55, 60, 70, 80, 90, or 100. In one embodiment, n=1, i.e., thelinker is (Gly4Ser) (SEQ ID NO: 196). In one embodiment, n=2, i.e., thelinker is (Gly4Ser)₂ (SEQ ID NO: 197). In another embodiment, n=3, i.e.,the linker is (Gly4Ser)₃ (SEQ ID NO: 198). In another embodiment, n=4,i.e., the linker is (Gly4Ser)₄ (SEQ ID NO: 199). In another embodiment,n=5, i.e., the linker is (Gly4Ser)₅ (SEQ ID NO: 200). In yet anotherembodiment, n=6, i.e., the linker is (Gly4Ser)₆ (SEQ ID NO: 202). Inanother embodiment, n=7, i.e., the linker is (Gly4Ser)₇ (SEQ ID NO:203). In yet another embodiment, n=8, i.e., the linker is (Gly4Ser)₈(SEQ ID NO: 204). In another embodiment, n=9, i.e., the linker is(Gly4Ser)₉ (SEQ ID NO: 205). In yet another embodiment, n=10, i.e., thelinker is (Gly4Ser)₁₀ (SEQ ID NO: 206).

Another exemplary Gly/Ser peptide linker comprises the amino acidsequence Ser(Gly4Ser)_(n) (SEQ ID NO: 201), wherein n is an integer thatis the same or higher than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 46, 50, 55, 60, 70, 80, 90, or 100. In one embodiment, n=1,i.e., the linker is Ser(Gly4Ser) (SEQ ID NO: 207). In one embodiment,n=2, i.e., the linker is Ser(Gly4Ser)₂ (SEQ ID NO: 208). In anotherembodiment, n=3, i.e., the linker is Ser(Gly4Ser)₃ (SEQ ID NO: 209). Inanother embodiment, n=4, i.e., the linker is Ser(Gly4Ser)₄ (SEQ ID NO:210). In another embodiment, n=5, i.e., the linker is Ser(Gly4Ser)₅ (SEQID NO: 211). In yet another embodiment, n=6, i.e., the linker isSer(Gly4Ser)₆ (SEQ ID NO: 212). In yet another embodiment, n=7, i.e.,the linker is Ser(Gly4Ser)₇ (SEQ ID NO: 213). In yet another embodiment,n=8, i.e., the linker is Ser(Gly4Ser)₈ (SEQ ID NO: 214). In yet anotherembodiment, n=9, i.e., the linker is Ser(Gly4Ser)₉ (SEQ ID NO: 215). Inyet another embodiment, n=10, i.e., the linker is Ser(Gly4Ser)₁₀ (SEQ IDNO: 216).

In certain embodiments, said Gly/Ser peptide linker can be insertedbetween two other sequences of the peptide linker (e.g., any of thepeptide linker sequences described herein). In other embodiments, aGly/Ser peptide linker is attached at one or both ends of anothersequence of the peptide linker (e.g., any of the peptide linkersequences described herein). In yet other embodiments, two or moreGly/Ser linkers are incorporated in series in a peptide linker. In oneembodiment, a peptide linker of the invention comprises at least aportion of an upper hinge region (e.g., derived from an IgG1, IgG2,IgG3, or IgG4 molecule), at least a portion of a middle hinge region(e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule) and a seriesof Gly/Ser amino acid residues (e.g., a Gly/Ser linker such as(Gly4Ser)_(n)) (SEQ ID NO: 195)).

A particular type of linker which can be present in an heterologousmoiety, for example an activatable clotting factor, is herein referredto as a “cleavable linker” which comprises a heterologousprotease-cleavage site (e.g., a factor XIa or thrombin cleavage site)that is not naturally occurring in the clotting factor and which caninclude additional linkers on either the N terminal of C terminal orboth sides of the cleavage site. Exemplary locations for such sitesinclude, e.g., placement between a heavy chain of a clotting factorzymogen and a light chain of a clotting factor zymogen.

Peptide linkers can be introduced into polypeptide sequences usingtechniques known in the art. Modifications can be confirmed by DNAsequence analysis. Plasmid DNA can be used to transform host cells forstable production of the polypeptides produced.

III.C Protease Cleavage Site

In some embodiments, a chimeric molecule can comprise a proteasecleavage site linking, for example, a light chain of a clotting factorzymogen and a heavy chain of the clotting factor zymogen (e.g., FVII). Aprotease-cleavage site linking a light chain of a clotting factorzymogen and a heavy chain of the clotting factor zymogen can be selectedfrom any protease-cleavage site known in the art. In one embodiment, theprotease-cleavage site is cleaved by a protease selected from the groupconsisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factorIXa, factor Xa, factor IIa (thrombin), and any combinations thereof. Theprotease-cleavage sites allow the light chain and the heavy chain of theclotting factor to be cleaved and dissociated from each other at thesite of injury. Exemplary FXIa cleavage sites include, e.g., KLTR (SEQID NO: 217), DFTR (SEQ ID NO: 218), TQSFNDFTR (SEQ ID NO: 219) andSVSQTSKLTR (SEQ ID NO: 220). Exemplary thrombin cleavage sites include,e.g., DFLAEGGGVR (SEQ ID NO: 221), TTKIKPR (SEQ ID NO: 222), LVPRG (SEQID NO: 223) and ALRPR (SEQ ID NO: 224).

In some embodiments, the protease-cleavage site can be combined with anintracellular processing site for efficient cleavage and activation. Forexample, an activatable clotting factor in the chimeric molecule cancomprise a heterodimer, which comprises a light chain of a clottingfactor associated with a heavy chain of the clotting factor by acovalent bond, wherein the N-terminus of the heavy chain of the clottingfactor is linked to a protease-cleavage site. The protease-cleavage sitecan be cleaved off at the site of coagulation, thus activating theclotting factor. Such constructs can be designed by inserting anintracellular processing site between the light chain of the clottingfactor zymogen and the protease-cleavage site, which is linked to theheavy chain of the clotting factor zymogen. The intracellular processingsite inserted therein can be processed (cleaved) by an intracellularprocessing enzyme upon expression in a host cell, thereby allowingformation of a zymogen-like heterodimer.

Examples of the intracellular processing enzymes include furin, a yeastKex2, PCSK1 (also known as PC1/Pc3), PCSK2 (also known as PC2), PCSK3(also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (alsoknown as PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 (also knownas PC7/LPC, PC8, or SPC7). Other processing sites are known in the art.In constructs that include more than one processing or cleavage site, itwill be understood that such sites can be the same or different.

IV. Methods of Preparation

The present disclosure also provides a nucleic acid molecule or a set ofnucleic acid molecules encoding (i) a GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein, or (ii) any of thechimeric molecules disclosed herein, or (iii) a complement thereof.

In one embodiment, the invention includes a nucleic acid moleculeencoding a polypeptide chain, which comprises a light chain of aclotting factor (e.g., FVII, FIX, or FX), a heterologous moiety (e.g., ahalf-life extending moiety), an intracellular processing site, a heavychain of the clotting factor (e.g., FVII, FIX, or FX), and a targetingmoiety which binds to a platelet (e.g., an anti-GPIIb/IIIa antibody orantigen-binding molecule thereof). In another embodiment, the nucleicacid molecule of the invention encodes a polypeptide chain comprising alight chain of a clotting factor (e.g., FVII, FIX, or FX), a targetingmoiety which binds to a platelet (e.g., an anti-GPIIb/IIIa antibody orantigen-binding molecule thereof), an intracellular processing site, aheavy chain of the clotting factor (e.g., FVII, FIX, or FX), and aheterologous moiety (e.g., a half-life extending moiety). In otherembodiments, the nucleic acid molecule encodes a polypeptide chaincomprising a light chain of a clotting factor (e.g., FVII, FIX, or FX),an intracellular processing site, a heavy chain of the clotting factor(e.g., FVII, FIX, or FX), a heterologous moiety (e.g., a half-lifeextending moiety), and a targeting moiety which binds to a platelet(e.g., an anti-GPIIb/IIIa antibody or antigen-binding molecule thereof).In some embodiments, the nucleic acid molecule encodes a polypeptidechain comprising a light chain of a clotting factor (e.g., FVII, FIX, orFX), an intracellular processing site, a heavy chain of the clottingfactor (e.g., FVII, FIX, or FX), a targeting moiety which binds to aplatelet (e.g., an anti-GPIIb/IIIa antibody or antigen-binding moleculethereof), and a heterologous moiety (e.g., a half-life extendingmoiety).

In some embodiments, the nucleic acid molecule comprises a set ofnucleotide sequences, a first nucleotide sequence encoding a firstpolypeptide chain comprising a light chain of a clotting factor (e.g.,FVII, FIX, or FX) and a heterologous moiety (e.g., a half-life extendingmoiety) and a second nucleotide sequence encoding a second polypeptidechain comprising a heavy chain of the clotting factor (e.g., FVII, FIX,or FX) and a targeting moiety which binds to a platelet (e.g., ananti-GPIIb/IIIa antibody or antigen-binding molecule thereof). In otherembodiments, the nucleic acid molecule comprises a set of nucleotidesequences, a first nucleotide sequence encoding a first polypeptidechain comprising a light chain of a clotting factor (e.g., FVII, FIX, orFX) and a targeting moiety which binds to a platelet (e.g., ananti-GPIIb/IIIa antibody or antigen-binding molecule thereof) and asecond nucleotide sequence encoding a second polypeptide chaincomprising a heavy chain of the clotting factor (e.g., FVII, FIX, or FX)and a heterologous moiety (e.g., a half-life extending moiety). In otherembodiments, the nucleic acid molecule comprises a set of nucleotidesequences, a first nucleotide sequence encoding a light chain of aclotting factor (e.g., FVII, FIX, or FX) and a second nucleotidesequence encoding a heavy chain of the clotting factor (e.g., FVII, FIX,or FX), a heterologous moiety (e.g., a half-life extending moiety), anda targeting moiety which binds to a platelet (e.g., an anti-GPIIb/IIIaantibody or antigen-binding molecule thereof). In some embodiments, thenucleic acid molecule comprises a set of nucleotide sequences, a firstnucleotide sequence encoding a light chain of a clotting factor (e.g.,FVII, FIX, or FX) and a second nucleotide sequence encoding a heavychain of the clotting factor (e.g., FVII, FIX, or FX), a targetingmoiety which binds to a platelet (e.g., an anti-GPIIb/IIIa antibody orantigen-binding molecule thereof), and a heterologous moiety (e.g., ahalf-life extending moiety).

Also provided are a vector or a set of vectors comprising such nucleicacid molecule or the set of the nucleic acid molecules or a complementthereof, as well as a host cell comprising the vector.

The instant disclosure also provides a method for producing a GPIIb/IIIaantibody or antigen-binding molecule thereof or chimeric moleculedisclosed herein, such method comprising culturing the host celldisclosed herein and recovering the antibody, antigen-binding moleculethereof, or the chimeric molecule from the culture medium.

A variety of methods are available for recombinantly producing aGPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein, or a chimeric molecule disclosed herein. It will be understoodthat because of the degeneracy of the code, a variety of nucleic acidsequences will encode the amino acid sequence of the polypeptide. Thedesired polynucleotide can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an earlier prepared polynucleotide.

Oligonucleotide-mediated mutagenesis is one method for preparing asubstitution, in-frame insertion, or alteration (e.g., altered codon) tointroduce a codon encoding an amino acid substitution (e.g., into aGPIIb/IIIa antibody variant). For example, the starting polypeptide DNAis altered by hybridizing an oligonucleotide encoding the desiredmutation to a single-stranded DNA template. After hybridization, a DNApolymerase is used to synthesize an entire second complementary strandof the template that incorporates the oligonucleotide primer. In oneembodiment, genetic engineering, e.g., primer-based PCR mutagenesis, issufficient to incorporate an alteration, as defined herein, forproducing a polynucleotide encoding a GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein, or any of thechimeric molecules disclosed herein.

For recombinant production, a polynucleotide sequence encoding apolypeptide (e.g., a GPIIb/IIIa antibody or antigen-binding moleculethereof disclosed herein, or any of the chimeric molecules disclosedherein) is inserted into an appropriate expression vehicle, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation.

The nucleic acid encoding the polypeptide (e.g., a GPIIb/IIIa antibodyor antigen-binding molecule thereof disclosed herein, or any of thechimeric molecules disclosed herein) is inserted into the vector inproper reading frame. The expression vector is then transfected into asuitable target cell which will express the polypeptide. Transfectiontechniques known in the art include, but are not limited to, calciumphosphate precipitation (Wigler et al. 1978, Cell 14:725) andelectroporation (Neumann et al. 1982, EMBO J. 1:841). A variety ofhost-expression vector systems can be utilized to express thepolypeptides described herein (e.g., a GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein, or any of thechimeric molecules disclosed herein) in eukaryotic cells. In oneembodiment, the eukaryotic cell is an animal cell, including mammaliancells (e.g., 293 cells, PerC6, CHO, BHK, Cos, HeLa cells). When thepolypeptide is expressed in a eukaryotic cell, the DNA encoding thepolypeptide (e.g., a GPIIb/IIIa antibody or antigen-binding moleculethereof disclosed herein, or any of the chimeric molecules disclosedherein) can also code for a signal sequence that will permit thepolypeptide to be secreted. One skilled in the art will understand thatwhile the polypeptide is translated, the signal sequence is cleaved bythe cell to form the mature chimeric molecule. Various signal sequencesare known in the art, e.g., native FVII signal sequence, native FIXsignal sequence, native FX signal sequence, native GPIIb signalsequence, native GPIIIa signal sequence, and the mouse IgK light chainsignal sequence. Alternatively, where a signal sequence is not included,the polypeptide (e.g., a GPIIb/IIIa antibody or antigen-binding moleculethereof disclosed herein, or any of the chimeric molecules disclosedherein) can be recovered by lysing the cells.

The GPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein, or any of the chimeric molecules disclosed herein can besynthesized in a transgenic animal, such as a rodent, goat, sheep, pig,or cow. The term “transgenic animals” refers to non-human animals thathave incorporated a foreign gene into their genome. Because this gene ispresent in germline tissues, it is passed from parent to offspring.Exogenous genes are introduced into single-celled embryos (Brinster etal. 1985, Proc. Natl. Acad. Sci. USA 82:4438). Methods of producingtransgenic animals are known in the art including transgenics thatproduce immunoglobulin molecules (Wagner et al. 1981, Proc. Natl. Acad.Sci. USA 78:6376; McKnight et al. 1983, Cell 34:335; Brinster et al.1983, Nature 306:332; Ritchie et al. 1984, Nature 312:517; Baldassarreet al. 2003, Theriogenology 59:831; Robl et al. 2003, Theriogenology59:107; Malassagne et al. 2003, Xenotransplantation 10: 267).

The expression vectors can encode for tags that permit for easypurification or identification of the recombinantly producedpolypeptide. Examples include, but are not limited to, vector pUR278(Ruther et al. 1983, EMBO J 2:1791) in which the polypeptide (e.g., aGPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein, or any of the chimeric molecules disclosed herein) codingsequence can be ligated into the vector in frame with the lac z codingregion so that a hybrid polypeptide is produced; pGEX vectors can beused to express proteins with a glutathione S-transferase (GST) tag.These proteins are usually soluble and can easily be purified from cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The vectors include cleavage sites, e.g.,for PreCission Protease (Pharmacia, Peapack, N.J.) for easy removal ofthe tag after purification.

For the purposes of this invention, numerous expression vector systemscan be employed. These expression vectors are typically replicable inthe host organisms either as episomes or as an integral part of the hostchromosomal DNA. Expression vectors can include expression controlsequences including, but not limited to, promoters (e.g.,naturally-associated or heterologous promoters), enhancers, signalsequences, splice signals, enhancer elements, and transcriptiontermination sequences. Preferably, the expression control sequences areeukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells. Expression vectors can also utilizeDNA elements which are derived from animal viruses such as bovinepapilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus,retroviruses (RSV, MMTV or MOMLV), cytomegalovirus (CMV), or SV40 virus.Others involve the use of polycistronic systems with internal ribosomebinding sites.

Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362). Cells which have integrated the DNA into their chromosomescan be selected by introducing one or more markers which allow selectionof transfected host cells. The marker can provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation.

An examplary expression vector is NEOSPLA (U.S. Pat. No. 6,159,730).This vector contains the cytomegalovirus promoter/enhancer, the mousebeta globin major promoter, the SV40 origin of replication, the bovinegrowth hormone polyadenylation sequence, neomycin phosphotransferaseexon 1 and exon 2, the dihydrofolate reductase gene and leader sequence.This vector has been found to result in very high level expression ofantibodies upon incorporation of variable and constant region genes,transfection in cells, followed by selection in G418 containing mediumand methotrexate amplification. Vector systems are also taught in U.S.Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated byreference in its entirety herein. This system provides for highexpression levels, e.g., >30 pg/cell/day. Other exemplary vector systemsare disclosed e.g., in U.S. Pat. No. 6,413,777.

In other embodiments, polypeptides of the invention (e.g., a GPIIb/IIIaantibody or antigen-binding molecule thereof disclosed herein, or any ofthe chimeric molecules disclosed herein) can be expressed usingpolycistronic constructs. In these expression systems, multiple geneproducts of interest such as multiple polypeptides of multimer bindingprotein can be produced from a single polycistronic construct. Thesesystems advantageously use an internal ribosome entry site (IRES) toprovide relatively high levels of polypeptides of the invention ineukaryotic host cells. Compatible IRES sequences are disclosed in U.S.Pat. No. 6,193,980 which is also incorporated herein. Those skilled inthe art will appreciate that such expression systems can be used toeffectively produce the full range of polypeptides disclosed in theinstant application.

More generally, once the vector or DNA sequence encoding a polypeptidehas been prepared, the expression vector can be introduced into anappropriate host cell. That is, the host cells can be transformed.Introduction of the plasmid into the host cell can be accomplished byvarious techniques well known to those of skill in the art. Theseinclude, but are not limited to, transfection (including electrophoresisand electroporation), protoplast fusion, calcium phosphateprecipitation, cell fusion with enveloped DNA, microinjection, andinfection with intact virus. See, Ridgway, A. A. G. “MammalianExpression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez andDenhardt, Eds. (Butterworths, Boston, Mass. 1988). Most preferably,plasmid introduction into the host is via electroporation. Thetransformed cells are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flowcytometry, immunohistochemistry, and the like.

As used herein, the term “transformation” refers in a broad sense to theintroduction of DNA into a recipient host cell that changes the genotypeand consequently results in a change in the recipient cell.

Along those same lines, “host cells” refers to cells that have beentransformed with vectors constructed using recombinant DNA techniquesand encoding at least one heterologous gene. In descriptions ofprocesses for isolation of polypeptides from recombinant hosts, theterms “cell” and “cell culture” are used interchangeably to denote thesource of polypeptide unless it is clearly specified otherwise. In otherwords, recovery of polypeptide from the “cells” can mean either fromspun down whole cells, or from the cell culture containing both themedium and the suspended cells.

In one embodiment, a host cell endogenously expresses an enzyme (or theenzymes) necessary to cleave a scFc linker (e.g., if such a linker ispresent and contains intracellular processing site(s)) during processingto form the mature polypeptide. During this processing, the scFc linkercan be substantially removed to reduce the presence of extraneous aminoacids. In another embodiment of the invention, a host cell istransformed to express one or more enzymes which are exogenous to thecell such that processing of a scFc linker occurs or is improved.

In one embodiment an enzyme which can be endogenously or exogenouslyexpressed by a cell is a member of the furin family of enzymes. CompletecDNA and amino acid sequences of human furin (i.e., PACE) were publishedin 1990. Van den Ouweland A M et al. (1990) Nucleic Acids Res. 18:664;Erratum in: Nucleic Acids Res. 18:1332 (1990). U.S. Pat. No. 5,460,950,issued to Barr et al., describes recombinant PACE and the coexpressionof PACE with a substrate precursor polypeptide of a heterologous proteinto improve expression of active, mature heterologous protein. U.S. Pat.No. 5,935,815, likewise describes recombinant human furin (i.e., PACE)and the coexpression of furin with a substrate precursor polypeptide ofa heterologous protein to improve expression of active, matureheterologous protein. Possible substrate precursors disclosed in thispatent include a precursor of Factor IX. Other family members in themammalian furin/subtilisin/Kex2p-like proprotein convertase (PC) familyin addition to PACE are reported to include PCSK1 (also known asPC1/Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin orPACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6),PCSK6 (also known as PACE4), or PCSK7 (also known as PC7/LPC, PC8, orSPC7). While these various members share certain conserved overallstructural features, they differ in their tissue distribution,subcellular localization, cleavage specificities, and preferredsubstrates. For a review, see Nakayama K (1997) Biochem J. 327:625-35.Similar to PACE, these proprotein convertases generally include,beginning from the amino terminus, a signal peptide, a propeptide (thatcan be autocatalytically cleaved), a subtilisin-like catalytic domaincharacterized by Asp, His, Ser, and Asn/Asp residues, and a Homo Bdomain that is also essential for catalytic activity and characterizedby an Arg-Gly-Asp (RGD) sequence. PACE, PACE4, and PC5 also include aCys-rich domain, the function of which is unknown. In addition, PC5 hasisoforms with and without a transmembrane domain; these differentisoforms are known as PC5B and PC5A, respectively. Comparison betweenthe amino acid sequence of the catalytic domain of PACE and the aminoacid sequences of the catalytic domains of other members of this familyof proprotein convertases reveals the following degrees of identity: 70percent for PC4; 65 percent for PACE4 and PC5; 61 percent for PC1/PC3;54 percent for PC2; and 51 percent for LPC/PC7/PC8/SPC7. Nakayama K(1997) Biochem J. 327:625-35.

PACE and PACE4 have been reported to have partially overlapping butdistinct substrates. In particular, PACE4, in striking contrast to PACE,has been reported to be incapable of processing the precursorpolypeptide of FIX. Wasley et al. (1993) J. Biol. Chem. 268:8458-65;Rehemtulla et al. (1993) Biochemistry. 32:11586-90. U.S. Pat. No.5,840,529, discloses nucleotide and amino acid sequences for human PC7and the notable ability of PC7, as compared to other PC family members,to cleave HIV gp160 to gp120 and gp41.

Nucleotide and amino acid sequences of rodent PC5 were first describedas PC5 by Lusson et al. (1993) Proc Natl Acad Sci USA 90:6691-5 and asPC6 by Nakagawa et al. (1993) J Biochem (Tokyo) 113:132-5. U.S. Pat. No.6,380,171 discloses nucleotide and amino acid sequences for human PC5A,the isoform without the transmembrane domain. The sequences of theseenzymes and method of cloning them are known in the art.

Genes encoding the polypeptides of the invention (e.g., a GPIIb/IIIaantibody or antigen-binding molecule thereof disclosed herein, or any ofthe chimeric molecules disclosed herein) can also be expressed innon-mammalian cells such as bacteria or yeast or plant cells. In thisregard it will be appreciated that various unicellular non-mammalianmicroorganisms such as bacteria can also be transformed; i.e., thosecapable of being grown in cultures or fermentation. Bacteria, which aresusceptible to transformation, include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the polypeptides typically become part ofinclusion bodies. The polypeptides must be isolated, purified and thenassembled into functional molecules.

In addition to prokaryates, eukaryotic microbes can also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141(1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. Thisplasmid already contains the TRP1 gene which provides a selection markerfor a mutant strain of yeast lacking the ability to grow in tryptophan,for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).The presence of the trpl lesion as a characteristic of the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Other yeast hosts such Pichia can also be employed. Yeast expressionvectors having expression control sequences (e.g., promoters), an originof replication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formethanol, maltose, and galactose utilization.

Alternatively, polypeptide-coding nucleotide sequences can beincorporated in transgenes for introduction into the genome of atransgenic animal and subsequent expression in the milk of thetransgenic animal (see, e.g., U.S. Pat. Nos. 5,741,957; 5,304,489; and5,849,992). Suitable transgenes include coding sequences forpolypeptides in operable linkage with a promoter and enhancer from amammary gland specific gene, such as casein or beta lactoglobulin.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.An affinity tag sequence (e.g. a His(6) tag) can optionally be attachedor included within the polypeptide sequence to facilitate downstreampurification.

Once expressed, the chimeric molecules can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)) and see specifically the methods used inthe instant Examples. Substantially pure proteins of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity mostpreferred, for pharmaceutical uses.

V. Methods of Use

The present disclosure also provides is a pharmaceutical compositioncomprising

(i) a GPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein;

(ii) a chimeric molecule disclosed herein;

(iii) a nucleic acid molecule or the set of nucleic acid moleculesdisclosed herein;

(iv) a vector or set of vectors disclosed herein; or

(v) any combinations thereof,

and a pharmaceutically acceptable carrier.

In some embodiments, administering (i) a chimeric molecule disclosedherein, (ii) a nucleic acid molecule or a set of nucleic acid moleculesdisclosed herein, (iii) a vector or a set of vectors disclosed herein,or (iii) a pharmaceutical composition disclosed herein, can be used, forexample, to reduce the frequency or degree of a bleeding episode in asubject in need, and/or reducing or preventing an occurrence of ableeding episode in a subject in need thereof. In some embodiments, thesubject has developed or has a tendency to develop an inhibitor againsttreatment with FVIII, FIX, or both. In some embodiments, the inhibitoragainst FVIII or FIX is a neutralizing antibody against FVIII, FIX, orboth.

In some embodiments, the bleeding episode can be caused by a bloodcoagulation disorder, for example, hemophilia A or hemophilia B. Inother embodiments, the bleeding episode can be derived fromhemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage intomuscles, oral hemorrhage, trauma, trauma capitis, gastrointestinalbleeding, intracranial hemorrhage, intra-abdominal hemorrhage,intrathoracic hemorrhage, bone fracture, central nervous systembleeding, bleeding in the retropharyngeal space, bleeding in theretroperitoneal space, bleeding in the illiopsoas sheath, or anycombinations thereof. In certain embodiments, the subject is a humansubject.

The instant disclosure also provides:

(a) a method to target a therapeutic or prophylactic agent (e.g., aclotting factor such as FVII) to the surface of platelets, wherein themethod comprises fusing the agent to one of the GPIIb/IIIa antibodies orantigen-binding molecules thereof disclosed herein;

(b) a method to increase the activity of a therapeutic or prophylacticagent (e.g., a clotting factor such as FVII) comprising fusing the agentto a GPIIb/IIIa antibody or antigen-binding molecule thereof disclosedherein; or,

(c) a method to improve the pharmacokinetic properties of a clottingfactor comprising fusing the clotting factor to the GPIIb/IIIa antibodyor antigen-binding molecule thereof disclosed herein.

In some embodiments, these methods further comprise fusing orconjugating the clotting factor and/or the GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein to a half-lifeextending moiety. In some embodiments, the therapeutic or prophylacticagent is FVII, FIX, or FX.

The present disclosure also provides a method of measuring the level ofplatelets in plasma of a subject in need thereof comprising contactingthe GPIIb/IIIa antibody or antigen binding molecule thereof disclosedherein with the plasma from the subject and measuring the level ofplatelets in plasma. This method can further comprise fusing orconjugating the clotting factor and/or the GPIIb/IIIa antibody orantigen-binding molecule thereof disclosed herein to a detectableheterologous moiety, for example, a fluorescent molecule or aradionuclide.

The invention also relates to a method of treating, ameliorating, orpreventing a hemostatic disorder to a subject comprising administering atherapeutically effective amount of a chimeric molecule of the inventionwhich comprises a clotting factor. The treatment, amelioration, andprevention by the chimeric molecule can be a bypass therapy. The subjectin the bypass therapy can have already developed an inhibitor to aclotting factor, e.g., FVIII or FIX, or is subject to developing aclotting factor inhibitor. Compositions for administration to a subjectinclude nucleic acid molecules which comprise a nucleotide sequenceencoding a chimeric molecule the invention.

In one embodiment, a chimeric molecule composition of the invention isadministered in combination with at least one other agent that promoteshemostasis. As an example, but not as a limitation, hemostatic agent caninclude FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, prothrombin, orfibrinogen or activated forms of any of the preceding. The clottingfactor or hemostatic agent can also include anti-fibrinolytic drugs,e.g., epsilon-amino-caproic acid, tranexamic acid.

In one embodiment, the composition (e.g., the polypeptide or nucleicacid molecule encoding the polypeptide) is one in which the clottingfactor is present in activatable form when administered to a subject.Such an activatable molecule can be activated in vivo at the site ofclotting after administration to a subject.

The chimeric molecule of the invention can be administeredintravenously, subcutaneously, intramuscularly, or via any mucosalsurface, e.g., orally, sublingually, buccally, sublingually, nasally,rectally, vaginally or via pulmonary route. The chimeric molecule can beimplanted within or linked to a biopolymer solid support that allows forthe slow release of the chimeric molecule to the desired site.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid for example a syrup or asuspension. The liquid can include suspending agents (e.g., sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil,oily esters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations can also include flavoring, coloring andsweetening agents. Alternatively, the composition can be presented as adry product for constitution with water or another suitable vehicle. Forbuccal and sublingual administration the composition can take the formof tablets, lozenges or fast dissolving films according to conventionalprotocols. For administration by inhalation, the chimeric molecules foruse according to the present invention are conveniently delivered in theform of an aerosol spray from a pressurized pack or nebulizer (e.g., inPBS), with a suitable propellant.

In one embodiment, the route of administration of the polypeptides ofthe invention is parenteral. The term parenteral as used herein includesintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal or vaginal administration. The intravenous form ofparenteral administration is preferred. While all these forms ofadministration are clearly contemplated as being within the scope of theinvention, a form for administration would be a solution for injection,in particular for intravenous or intraarterial injection or drip.Usually, a suitable pharmaceutical composition for injection cancomprise a buffer (e.g., acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g.,human albumin), etc. However, in other methods compatible with theteachings herein, the polypeptides can be delivered directly to the siteof the adverse cellular population thereby increasing the exposure ofthe diseased tissue to the therapeutic agent.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1M and preferably 0.05Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives can also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., a polypeptide by itself or incombination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations can be packaged and sold in the form of a kit. Sucharticles of manufacture will preferably have labels or package insertsindicating that the associated compositions are useful for treating asubject suffering from, or predisposed to clotting disorders.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

Effective doses of the compositions of the present invention, for thetreatment of conditions vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human but non-human mammalsincluding transgenic mammals can also be treated. Treatment dosages canbe titrated using routine methods known to those of skill in the art tooptimize safety and efficacy.

In one embodiment, the dose of a biologically active moiety (e.g.,comprising FVII), can range from about 90 to 270 μg/kg or 0.090 to 0.270mg/kg. In another embodiment, the dose of a biologically active moiety(e.g., comprising FX), can range from about 1 μg/kg to 400 mg/kg.

Dosages can range from 1000 μg/kg to 0.1 ng/kg body weight. In oneembodiment, the dosing range is 1 ug/kg to 100 μg/kg. The protein can beadministered continuously or at specific timed intervals. In vitroassays can be employed to determine optimal dose ranges and/or schedulesfor administration. In vitro assays that measure clotting factoractivity are known in the art, e.g., STA-CLOT Vlla-rTF clotting assay.Additionally, effective doses can be extrapolated from dose-responsecurves obtained from animal models, e g., a hemophiliac dog (Mount etal. 2002, Blood 99: 2670).

Doses intermediate in the above ranges are also intended to be withinthe scope of the invention. Subjects can be administered such dosesdaily, on alternative days, weekly or according to any other scheduledetermined by empirical analysis. An exemplary treatment entailsadministration in multiple dosages over a prolonged period, for example,of at least six months. In some methods, two or more polypeptides can beadministered simultaneously, in which case the dosage of eachpolypeptide administered falls within the ranges indicated.

Polypeptides of the invention can be administered on multiple occasions.Intervals between single dosages can be daily, weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of modified polypeptide or antigen in the patient. Alternatively,polypeptides can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the polypeptide in thepatient.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the polypeptides of the inventionor a cocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance or minimize effects ofdisease. Such an amount is defined to be a “prophylactic effectivedose.” A relatively low dosage is administered at relatively infrequentintervals over a long period of time. Some patients continue to receivetreatment for the rest of their lives.

Polypeptides of the invention can optionally be administered incombination with other agents that are effective in treating thedisorder or condition in need of treatment (e.g., prophylactic ortherapeutic).

As used herein, the administration of polypeptides of the invention inconjunction or combination with an adjunct therapy means the sequential,simultaneous, coextensive, concurrent, concomitant or contemporaneousadministration or application of the therapy and the disclosedpolypeptides. Those skilled in the art will appreciate that theadministration or application of the various components of the combinedtherapeutic regimen can be timed to enhance the overall effectiveness ofthe treatment. A skilled artisan (e.g., a physician) would be readily beable to discern effective combined therapeutic regimens without undueexperimentation based on the selected adjunct therapy and the teachingsof the instant specification.

It will further be appreciated that the polypeptides of the instantinvention can be used in conjunction or combination with an agent oragents (e.g., to provide a combined therapeutic regimen). Exemplaryagents with which a polypeptide of the invention can be combined includeagents that represent the current standard of care for a particulardisorder being treated. Such agents can be chemical or biologic innature. The term “biologic” or “biologic agent” refers to anypharmaceutically active agent made from living organisms and/or theirproducts which is intended for use as a therapeutic.

The amount of agent to be used in combination with the polypeptides ofthe instant invention can vary by subject or can be administeredaccording to what is known in the art. See for example, Bruce A Chabneret al., Antineoplastic Agents, in Goodman & Gilman's The PharmacologicalBasis of Therapeutics 1233-1287 ((Hardman et al., eds., 9th ed. 1996).In another embodiment, an amount of such an agent consistent with thestandard of care is administered.

As previously discussed, the polypeptides of the present invention, canbe administered in a pharmaceutically effective amount for the in vivotreatment of clotting disorders. In this regard, it will be appreciatedthat the polypeptides of the invention can be formulated to facilitateadministration and promote stability of the active agent. Preferably,pharmaceutical compositions in accordance with the present inventioncomprise a pharmaceutically acceptable, non-toxic, sterile carrier suchas physiological saline, non-toxic buffers, preservatives and the like.Of course, the pharmaceutical compositions of the present invention canbe administered in single or multiple doses to provide for apharmaceutically effective amount of the polypeptide.

In one embodiment, a chimeric molecule of the invention is administeredas a nucleic acid molecule. Nucleic acid molecules can be administeredusing techniques known in the art, including via vector, plasmid,liposome, DNA injection, electroporation, gene gun, intravenouslyinjection or hepatic artery infusion. Vectors for use in gene therapyembodiments are known in the art.

In keeping with the scope of the present disclosure, the chimericmolecule of the invention can be administered to a human or other animalin accordance with the aforementioned methods of treatment in an amountsufficient to produce a therapeutic or prophylactic effect.

The chimeric molecules of the invention have many uses as will berecognized by one skilled in the art, including, but not limited tomethods of treating a subject with a disease or condition. The diseaseor condition can include, but is not limited to, hemostatic disorders.

In one embodiment, the invention relates to a method of treating asubject having a hemostatic disorder comprising administering atherapeutically effective amount of at least one chimeric molecule ofthe invention.

The chimeric molecules of the invention treat or prevent a hemostaticdisorder by promoting the formation of a fibrin clot. The chimericmolecule of the invention can activate any member of a coagulationcascade. The clotting factor can be a participant in the extrinsicpathway, the intrinsic pathway or both. A chimeric molecule of theinvention can be used to treat hemostatic disorders, e.g., those knownto be treatable with the particular clotting factor present in thechimeric molecule. The hemostatic disorders that can be treated byadministration of the chimeric molecule of the invention include, butare not limited to, hemophilia A, hemophilia B, von Willebrand'sdisease, Factor XI deficiency (PTA deficiency), Factor XII deficiency,as well as deficiencies or structural abnormalities in fibrinogen,prothrombin, Factor V, Factor VII, Factor X, or Factor XIII.

In one embodiment, the hemostatic disorder is an inherited disorder. Inone embodiment, the subject has hemophilia A, and the chimeric moleculecomprises activated or protease-activatable FVII linked to or associatedwith a GPIIb/IIIa antibody or antigen-binding molecule thereof and ahalf-life extending heterologous moiety. In another embodiment, thesubject has hemophilia A and the chimeric molecule comprises activatedor protease-activatable FVII linked to or associated with a GPIIb/IIIaantibody or antigen-binding molecule thereof and a half-life extendingheterologous moiety. In other embodiments, the subject has hemophilia Band the chimeric molecule comprises activated or protease-activatableFVII or FX linked to or associated with a GPIIb/IIIa antibody orantigen-binding molecule thereof and a half-life extending heterologousmoiety. In some embodiments, the subject has inhibitory antibodies toFVIII or FVIIIa and the chimeric molecule comprises activated orprotease-activatable FVII linked to or associated with a GPIIb/IIIaantibody or antigen-binding molecule thereof and a half-life extendingheterologous moiety. In yet other embodiments, the subject hasinhibitory antibodies against FIX or FIXa and the chimeric moleculecomprises activated or protease-activatable FVII linked to or associatedwith a GPIIb/IIIa antibody or antigen-binding molecule thereof and ahalf-life extending heterologous moiety. In still other embodiments, thesubject has inhibitory antibodies to FVIII or FVIIIa and the chimericmolecule comprises activated or protease-activatable FX linked to orassociated with a GPIIb/IIIa antibody or antigen-binding moleculethereof and a half-life extending heterologous moiety. In certainembodiments, the subject has inhibitory antibodies against FIX or FIXaand the chimeric molecule comprises activated or protease-activatable FXlinked to or associated with a GPIIb/IIIa antibody or antigen-bindingmolecule thereof and a half-life extending heterologous moiety.

Chimeric molecules of the invention comprising a clotting factor (e.g.,FVII) can be used to prophylactically treat a subject with a hemostaticdisorder. Chimeric molecules of the invention comprising a clottingfactor (e.g., FVII) can be used to treat an acute bleeding episode in asubject with a hemostatic disorder.

In one embodiment, the hemostatic disorder is the result of a deficiencyin a clotting factor, e.g., FVII, FIX, or FVIII. In another embodiment,the hemostatic disorder can be the result of a defective clottingfactor. In another embodiment, the hemostatic disorder can be anacquired disorder. The acquired disorder can result from an underlyingsecondary disease or condition. The unrelated condition can be, as anexample, but not as a limitation, cancer, an autoimmune disease, orpregnancy. The acquired disorder can result from old age or frommedication to treat an underlying secondary disorder (e.g. cancerchemotherapy).

The invention also relates to methods of treating a subject who does nothave a hemostatic disorder or a secondary disease or condition resultingin acquisition of a hemostatic disorder. The invention thus relates to amethod of treating a subject in need of a general hemostatic agentcomprising administering a therapeutically effective amount of at leastone chimeric molecule of the invention. For example, in one embodiment,the subject in need of a general hemostatic agent is undergoing, or isabout to undergo, surgery. The chimeric molecule of the invention can beadministered prior to or after surgery as a prophylactic. The chimericmolecule of the invention can be administered during or after surgery tocontrol an acute bleeding episode. The surgery can include, but is notlimited to, liver transplantation, liver resection, or stem celltransplantation. In another embodiment, the chimeric molecule of theinvention can be used to treat a subject having an acute bleedingepisode who does not have a hemostatic disorder. The acute bleedingepisode can result from severe trauma, e.g., surgery, an automobileaccident, wound, laceration gun shot, or any other traumatic eventresulting in uncontrolled bleeding.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention. All patents and publicationsreferred to herein are expressly incorporated by reference in theirentireties.

EXAMPLES General Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, biophysics,molecular biology, recombinant DNA technology, immunology (especially,e.g., antibody technology), and standard techniques in electrophoresis.See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: ColdSpring Harbor Laboratory Press (1989); Antibody Engineering Protocols(Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996);Antibody Engineering: A Practical Approach (Practical Approach Series,169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual,Harlow et al., CS.H.L. Press, Pub. (1999); and Current Protocols inMolecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

Example 1 Identification and Characterization of Platelet-TargetedAntibodies

A summary of the process used for production, identification, andcharacterization of antibodies against GPIIb/IIIa disclosed herein, andconstructs derived from these antibodies is presented in FIG. 1.

Hybridomas were generated from BALB/C mice immunized with plasmidscontaining DNA sequences encoding GPIIb/IIIa (SEQ ID NOs: 183 and 184)according to methods known in the art. Hybridomas were then screened forbinding to human and cynomolgus monkey platelets using flow cytometry,and for binding to GPIIb/IIIa using Enzyme-linked immunosorbent assays(ELISA). To determine binding to human and money platelets, gel-purifiedhuman or monkey (cynomolgus) platelets in Tyrode's buffer were incubatedwith hybridoma supernatant. Following a 30 minute incubation, cells werefixed in 1% formaldehyde. Following fixation, cells were washed inTyrode's buffer and a detection antibody was added (JacksonImmunoresearch goat anti-mouse IgG-PE conjugated). Antibody binding wasdetected by flow cytometry.

The binding of supernatants from hybridomas to human GPIIb/IIIa (αIIbβ)was determined by using ELISA as follows. Costar plates (Cat. No. 3590)were coated with 100 μl/well of 5 μg/mL human GPIIb/IIIa (Calbiochem CatNo. 528240) in measuring buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mMMgCl₂, and 1 mM MnCl₂) and incubated for 1 hour at 37° C. with shaking.Wells were washed three times with TBST using a plate washer. Blockingwas performed using 200 μl of measuring buffer containing 5% BSA (BovineSerum albumin, Jackson Cat No 001 000 173) per well, and incubating 1hour at 37° C. with shaking. 100 μL of hybridoma supernatant were addedassay wells, incubated for 1 hour at 37° C. with shaking, and washedthree times with TBST. A 1:10,000 dilution of goat anti mouse IgGHRP(Southern Biotech (Cat. No. 1010 05) in measuring buffer was added,incubated for 1 hour at 37° C. with shaking, and washed three times withTBST. HRP presence was developed using TMB and O.D. read at 450 nm usinga Molecular Devices plate reader.

The supernatants from hybridomas which tested positive in the ELISAassays were mixed with platelets and screened for platelet activationusing flow cytometry as follows.

(a) Reagents: Citrated human whole blood; Sepharose 2B beads (GEHealthcare); Tyrode's buffer with 1 mg/mL BSA (no calcium); Tyrode'sbuffer with 5 mM CaCl₂) and 1 mg/mL BSA; 32% paraformaldehyde (PFA) (EMSciences); PAC1 FITC antibody (BD Cat. No. 340507); CD62 PE antibody (BDCat. No. 555524); ADP; SFFLRN peptide (Anaspec, Cat. No. 24191); IV.3Fabb anti CD32 (StemCell, Cat. No. 01470).

(b) Platelet purification: A 10 mL Sepharose 2B bead column was packedand equilibrated with 30 mL of Tyrode's buffer containing 1 mg/mL BSA. Avolume of 1 to 1.5 mL of platelet-rich plasma (PRP) was loaded onto theequilibrated Sepharose column and allowed to enter the packed beads bygravity, followed with approximately 5 mL of Tyrode's buffer. The turbiddrops, which contained the platelets, were collected.

(c) Assay: First, 50 μL aliquots of hybridoma supernatant were added toassay wells of a 96 well round bottom plate. 10 μL of PAC1 FITC and 10μL of CD62PE were added to all control and assay wells. 10 μL of ADP and10 μL of SFFLRN were added to all control wells (no hybridomasupernatant). 10 μL of IV.3 inhibitor (antibody to FcγRIIA) were addedwells to see if activation was Fc or antibody mediated. Next, a 50 μLaliquot of concentrated resting platelets, which was purified asdescribed above, was added to all wells. Plates were incubated for 30minutes in the dark and at room temperature. Cells were fixed with 1%PFA (final concentration) for 10 minutes at room temperature (a volumeof 2% PFA equal to the content of each well was added). After fixation,samples were analyzed by flow cytometry.

The antibodies that did not activate platelets upon binding toGPIIb/IIIa (see FIG. 2B) were selected as candidates for clotting factortargeting moieties. The antibodies that activate platelets upon bindingto GPIIb/IIIa were excluded from selection (see FIG. 2C)

Antibodies can also activate platelets by binding to the FcγRIIAreceptor via the Fc region (see FIG. 2D), which were not excluded fromthe selection because their antigen binding portion contain no Fc regionand therefore not bind to the FcγRIIA receptor. These antibodies can beidentified by blocking the FcγRIIA receptor with an inhibitor.

FIG. 3 shows flow cytometry profiles from platelet activation assays inwhich CD62p and PAC1 were detected (FIG. 3A). CD62p, also known asP-selectin, is expressed on the platelet surface upon plateletactivation. PAC1 is a pentameric IgM that binds to agonist-stimulatedplatelets, and PAC1 binding is therefore indicative ofGPIIb/IIIa-mediated activation. Experiments were performed in theabsence and presence of an FcγRIIA receptor inhibitor (IV.3 Fab; seeNaik et al., Biochem. J. 310:155-162 (1995)) in order to detectantibodies capable of activating platelets indirectly via simultaneousbinding to GPIIb/IIIa and to the FcγRIIA receptor (see FIGS. 3B, 3C, 3D,and 3E).

FIG. 3B shows flow cytometry profiles observed when only cell culturemedium was present, and when ADP and a PAR1 platelet receptor agonistpeptide with amino sequence SFLLRN (SEQ ID NO: 225) were present. FIG.3C shows flow cytometry profiles corresponding to an antibody capable ofactivating platelets both in the presence and absence of the FcγRIIAinhibitor monoclonal antibody. Thus, the antibody activates platelets bydirect binding to GPIIb/IIIa. FIG. 3D shows flow cytometry profilescorresponding to an antibody not capable of activating platelets both inthe presence and absence of the FcγRIIA inhibitor monoclonal antibody.FIG. 3E shows flow cytometry profiles corresponding to antibodiesindirectly activating platelets, i.e., the antibodies bindsimultaneously to GPIIa/IIIb and to the FcγRIIA receptor.

The supernatants from non-activating hybridomas were subject toadditional characterization assays (i) to confirm antibody binding tohuman and cynomolgus platelets, (ii) to determine antibody bindingspecificity for the α and/or β subunit of GPIIb/IIIa, and (iii) todetermine whether the antibodies can compete with fibrinogen for bindingto platelets. Fibrinogen is the natural ligand of GPIIb/IIIa and itsbinding to GPIIbIIIa is essential to mediate platelet aggregation. Thus,the antibodies that compete with the binding of fibrinogen to GPIIb/IIIawere excluded from the selection.

Antibody binding to the α and/or β subunit of GPIIb/IIIa was assessedusing ELISA, whereas antibody competition with fibrinogen was assessedusing flow cytometry. Antibodies determined to be non-activating (e.g.,clones 34D10, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 12B2,38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, 13A1) were clustered into 6different groups according to the VH domain sequence similarity, α or βsubunit specificity, ability to compete with fibrinogen, and relativestrength of the signals measured via ELISA and flow cytometry (see TABLE1).

TABLE 1 Platelet Specific Non-Activating Anti-GPIIb/IIIa MonoclonalAntibodies Group/Antibody Antibody/ SEQ CDR SEQ IDs CharacteristicsChain ID CDR1 CDR2 CDR3 1/α subunit specific 2A2 HC 3 25 26 27 Nofibrinogen competition LC 4 28 29 30 34D10^(#) HC 1 31 32 33 LC 2 34 3536 35D1 HC 97 111 112 113 LC 98 114 115 116 36A8 HC 5 37 38 39 LC 6 4041 42 4B11 HC 7 43 44 45 LC 99 117 118 119 2/α subunit specific. 1H6 HC8 46 47 48 Fibrinogen competition LC 9 49 50 51 38G8 HC 100 120 121 122LC 101 123 124 125 21F10 HC 102 126 127 128 LC 103 129 130 131 38A8 HC10 52 53 54 LC 11 55 56 57 3/α subunit specific. 18F7 HC 12 58 59 60Fibrinogen competition. Low LC 13 61 62 63 ELISA signal v flow cytometry4/β subunit specific. No 12B2^($) HC 14 64 65 66 fibrinogen competitionLC 15 67 68 69 38F6 HC 16 70 71 72 LC* 104 132 133 134 13C1 HC 105 135136 137 LC* 106 138 139 140 5/β subunit specific. 5C4 HC 17 73 74 75Fibrinogen competition LC* 107 141 142 143 23C10 HC 18 76 77 78 LC* 108144 145 146 37C7 HC 109 147 148 149 LC* 110 150 151 152 28C2 HC 19 79 8081 LC 20 82 83 84 9D6 HC 21 85 86 87 9D6 LC 22 88 89 90 6/β subunitspecific. 28F4 HC 23 91 92 93 Fibrinogen competition. Low LC 24 94 95 96ELISA signal v flow cytometry *= Not sequenced. HC = Heavy Chain (i.e.,VH). LC = Light Chain (i.e., VL) ^(#)α subunit or complex specific ^($)βsubunit specific

Several non-activating antibodies identified using the screening methoddescribed above shared the same VH or VL domains, as show in FIG. 4. Forexample, 35D1 and 34D10 shared the same VH domain.

Unique VH and VL sequences were aligned using the ClustalX program usingstandard parameters (FIG. 5). Sequence designators in FIG. 5, which arealso used in the sequence alignments of FIGS. 6 to 12, follow the schema“SEQ ID NO-Antibody-BindingDomain.” Accordingly “SEQ22-9D6LC”corresponds to SEQ ID NO:22, which is the Light Chain variable domain(i.e., VL) of the 9D6 antibody. Similarly, “SEQ08-1H6HC” corresponds toSEQ ID NO:8, which is the Heavy Chain variable domain (i.e., VH) of the1H6 antibody.

The character “*” in the multiple sequence alignments indicatespositions which have a single, fully conserved amino acid residue. Thecharacter “:” indicates that one strong conservation group is fullyconserved. Strong conservation amino acid groups are STA, NEQK, NHQK,NDEQ, QHRK, MILV, MILF, HY, and FYW. The character “.” indicates thatone weaker conservation group is fully conserved. Weaker conservationamino acid groups are CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK,NEQHRK, FVLIM, and HFY. Amino acids are shaded in FIG. 5 according tothe rules set out below. Rules are specified according to the formula:(A, C, and D): {50%, P,Q,RSTV} {85%, W,Y}, where amino acid residue orresidues in the alignment column are given first in the round brackets.More than one amino acid can be specified between the round brackets, inwhich case the rules apply to each of these amino acid residues. Next,the rule or rules to assign a given color are provided between curlybraces. Only one rule has to be met for the color to be applied. Theminimum percentage of occurrence in the alignment column is given first,followed by the amino acid residue or residues which must meet or exceedthis percentage within the column. If a group of amino acid residues isconcatenated together, such as “RSTV,” then any combination of theseresidues in total must meet or exceed the given percentage for the colorto be applied. For amino acid residues or residue groups separated bycommas, at least one of these must by itself exceed the percentage.

Highlighting (shading) rules in FIG. 5 are based on conservation andcommon physicochemical and/or structural properties. For example, tinyresidues (G) and prolines (P) are always highlighted when present. Thefollow highlighting rules were applied:

-   -   W, L, V, I, M, F, A, and C amino acid residues are highlighted        according to the following set of rules: (W, L, V, I, M, F):        {50%, P} {60%, WLVIMAFCYHP}; (A): {50%, P} {60%, WLVIMAFCYHP}        {85%, T, S, G}; and (C): {50%, P} {60%, WLVIMAFCYHP} {85%, S}.    -   K and R amino acid residues are highlighted according to the        following set of rules: (K, R): {60%, KR} {85%, Q}.    -   T, S, N, and Q amino acid residues are highlighted according to        the following set of rules: (T): {50%, TS} {60%, WLVIMAFCYHP};        (S): {50%, TS} {80%, WLVIMAFCYHP}; (N): {50%, N} {85%, D}; and,        (Q): {50%, QE)} {60%, KR}.    -   C amino acid residues are highlighted according to the following        rule: (C): {85%, C}.    -   D and E amino acid residues are highlighted according to the        following set of rules: (D): {50%, DE, N}; and (E): {50%, DE,        QE}.    -   G amino acid residues are highlighted in according to the        following rule: (G): {Always}.    -   H and Y amino acid residues highlighted according to the        following set of rules: (H,Y): {50%, P} {60%, WLVIMAFCYHP}.    -   P amino acid residues are highlighted according to the following        rule: (P): {Always}.

Multiple sequence alignments corresponding to the VH and VL domains ofthe above identified antibodies are shown in FIG. 6 and FIG. 7,respectively. The multiple sequence alignments show the location of thecomplementarity determining regions CDR1, CDR2, and CDR3 in the VH andVL domain and their location according to the EU numbering system(Kabat, E. A., Wu, T. T., Perry, H., Gottesman, K., and Foeller, C.(1991) “Sequences of Proteins of Immunological Interest,” 4th ed., U.S.Govt. Printing Off. No. 165-492, Nethesda, Md.).

FIG. 8 shows percent identity matrices showing the percentage ofsequence identity among each pair of the VH and VL sequences shown inthe multiple sequence alignments of FIGS. 6 and 7. Column designationscorrespond to the designation applied to each row in the matrix. Forexample, column 2 corresponds to “2: SEQ 22-9D6LC.” The sequencesaligned in FIG. 6 and FIG. 7 were clustered according to the specificityof the antibodies for the at or β subunits of GPIIb/IIIa (see FIG. 9 andFIG. 10), and according to their ability to compete with fibrinogen forbinding to GPIIb/IIIa (see FIG. 11 and FIG. 12).

Example 2 Platelet-Targeted Chimeric Molecules ComprisingAnti-GPIIb/IIIa Antibodies and FVIIa

The above described monoclonal antibodies against GPIIb/IIIa were usedto target the FVIIa clotting factor to the surface of platelets.Accordingly, scFv's derived from the platelet-specific monoclonalantibodies identified according to the methods disclosed in Example 1were recombinantly fused to FVIIa using molecular biology methods knownin the art. In the resulting chimeric molecules, the C-terminus of theheavy chain of FVIIa was fused to the N-terminus of an scFv comprising aVH and a VL domain derived from non-activating platelet-targetingantibodies identified in Example 1 (see FIG. 13). A chimeric moleculecomprising an scFv derived from the GPIIb/IIIa atsubunit/complex-specific 34D10 antibody was designated “FVII-189”. Asecond chimeric molecule comprising an scFv derived from the GPIIb/IIIaβ subunit-specific 12B2 monoclonal antibody was designated “FVII-206”. Achimeric molecule comprising an scFv from a known antibody (PDG13) wasdesignated as “FVII-163”. A chimeric molecule containing an scFV from38A8 monoclonal antibody was designated as “FVII-204”. Theseplatelet-targeted FVIIa variants were characterized for their affinityto GPIIb/IIIa receptor, their ability to bind to platelets, theirprocoagulant activity, their effect on platelet activation and plateletfunction, and their effect on platelet clearance in animals.

The affinity of platelet-targeted FVII-189, FVII-206, FVII-163 chimericmolecules to GPIIb/IIIa receptor was measured using surface plasmonresonance (SPR). GPIIb/IIIa was modified to incorporate a tag for invivo biotinylation, and an HPC4 tag for purification (see FIG. 14). TheGPIIb/IIIa protein were transiently expressed in HEK293 cells andsubsequently purified. GPIIb/IIIa nanodiscs were formed by firstcombining the GPIIb/IIIa protein with the scaffold protein,phospholipids, and detergent, followed with the removal of the detergent(FIG. 14). (see, Zhu et al., Sci. Transl. Med. 4, 125ra32 (2012), whichis hereby incorporated by reference in its entirety).

The nanodiscs, which contained the biotinylated GPIIb/IIIa receptor,were bound to BIACORE® streptavidin chips, and binding of the FVII-189,FVII-206, and FVII-163 chimeric molecules was measured using SPR (seeFIG. 15 and TABLE 2).

TABLE 2 Affinity of FVII-189 and FVII-206 Chimeric Molecules toGPIIb/IIIa receptor as measured by SPR FVIIa Variant Antibody (scFv)Moiety Affinity (nM) FVII-189 34D10 5.2 FVII-206 12B2 5.3 FVII-163 PDG138.8 Recombinant FVIIa None No binding observed

SPR results indicate that both x-targeted and 3-targeted chimericmolecules bound to GPIIb/IIIa with similar affinity. Compared to theknown antibody (PDG13), the 34D10 and 12B2 antibodies provide strongerGPIIb/IIIa affinity to FVIIa.

The ability of the FVIIa chimeric molecules to bind to platelets wasmeasured in a flow cytometry-based platelet-binding assay. Briefly, theFVIIa chimeric molecules were spiked into a citrated human whole bloodand incubated for 30 mins. The blood was then stained with anAPC-conjugated anti-human CD42b antibody and a FITC-conjugated anti-FVIIpolyclonal antibody to label the platelets and FVII protein,respectively. After fixing with paraformadyhyde, the stained blood wasanalyzed by flow cytometry. The platelets were identified by scattergating followed by APC-fluorescence gating; and relative FVIIconcentration was represented by the median fluorescent value ofFITC-fluorescence gating on gated platelets. As shown in FIG. 18, allplatelet-targeted FVII chimeric molecules described above (FVII-163,FVII-189, FVII-204, and FVII-206) were capable of binding to humanplatelets. No binding was observed to human platelets for recombinantFVIIa alone (in the absence of a targeting moiety). See also Table 2.

The procoagulant activity of the platelet-targeted FVIIa chimericmolecules was measured using a FVIIa specific soluble tissue factorprothrombin time (sTF-PT) and rotational thromboelastometry (ROTEM)assays. The specific activity of the platelet-targeted rFVIIa variantsFVII-189, FVII-206, and FVII-204, as measured by sTF-PT assays, wasfound to be comparable to that of rFVIIa, indicating that linking of theplatelet-targeting moiety does not affect rFVIIa's catalytic activity.In the presence of platelets, such as the ROTEM assays using the wholeblood from human Hemophila A donors, the platelet-targeted rFVIIavariants showed improved clotting activities. Specifically, FVII-189 andFVII-206 displayed a 25-50 fold increase in activity compared to rFVIIa(FIGS. 16A,16B, 17B). Improvement in activity was also observed inFVII-204 (FIG. 17A).

Platelet activation was measured using flow cytometry as described inExample 1. FIG. 19 shows that platelets were not activated by either theplatelet-targeted FVII-189 chimeric molecule (targeting the αchain/complex) or the platelet-targeted FVII-206 chimeric molecule(targeting the β chain). FVII-130 is a platelet-targeted chimericmolecule known to activate platelets and was used as a positive controlusing the same experimental conditions (see FIG. 19).

FVII-189 (FIG. 20A) and FVII-206 (FIG. 20B) did not inhibit ADP-inducedplatelet aggregation in platelet-rich plasma, indicating that none ofthese chimeric molecules inhibited platelet function. REOPRO®(abciximab), a monoclonal antibody that blocks the pathway to plateletaggregation, was used as a control. Accordingly, platelet-targetedchimeric molecules comprising targeting moieties derived from theanti-GPIIb/IIIa antibodies disclosed herein did not activate or inhibitplatelet function.

The effect of platelet-targeting rFVIIa chimeric molecules on plateletclearance in vivo was investigated in the NOD SCID gamma (NSG) mice withcirculating human platelets since none of the targeting moieties iscapable of binding mouse platelets. Human platelet-rich plasma (PRP) wasprepared from the citrated whole blood. The PRP was concentrated andadministrated via retro-obital into NSG mice. After 30 minutes, theclotting factors were dosed at 5 nmol/kg by tail-vein injection, and theblood samples were collected via tail laceration at 5 minutes before,and at various times after clotting factor dosing. To quantify the humanplatelets, the blood was stained with a cocktail containingPE-conjugated anti-mouse CD61, APC-conjugated anti-human CD42b tovisualize by flow cytometry the mouse platelets and human platelet,respectively. The relative human platelet counts was obtained by thescattering gating for platelets, and the PE, APC fluorescent gating formouse and human platelets, respectively. As shown in FIG. 21, a controlprotein FVII-130, which is known to promote platelet clearance and causethrombocytopenia, led a rapid human platelet clearance in this model;more than 90% of injected human platelets were cleared within 1 hourfollowing the administration of FVII-130. On the contrary, no effect onhuman platelet clearance can be detected when treated the mice withFVII-189, FVII-206, and FVII-204, indicating that theseplatelet-targeted rFVIIa chimeric molecules do not affect the humanplatelet clearance in vivo.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternative boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The present application claims benefit to U.S. Provisional ApplicationNo. 61/827,165, filed May 24, 2014, which is incorporated herein byreference in its entirety.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein.

All patents and publications cited herein are incorporated by referenceherein in their entirety.

SEQUENCE LISTING:  >SEQ_ID_NO: 1 34D10 HC EVKLVESGGGLVKPGGSLKLSCAASGFTFSAYAMSWVRQTPEKRLEWVASISSGGTTYYPDSVKRRFTIS RDNARNILYLQMSSLRSEDTAMYYCTRGGDYGYALDYWGQGTSVTVSS  >SEQ_ID_NO: 2 34D10 LC ENVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSY SLTISSMEGEDAATYYCQQFSSSPWTFGGGTKLEIKR  >SEQ_ID_NO: 3 2A2 HC EVKLVESGGGLVKPGGSLKLSCAASGFTFRTYAMSWVRQTPEKRLEWVASISSGSSTYYLDSVKGRFTIS RDNARNILYLQMSSLRSEDTAMYYCARGGDYGYALDYWGQGTSVTVSS  >SEQ_ID_NO: 4 2A2 LC ENVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQQKSDASPKLWIYYTSNLAPGVPTRFSGSGSGNSY SLTISSLEGEDAGTYYCQQFSSSPWTFGGGTKLEIKR  >SEQ_ID_NO: 5 36A8 HC EVRLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASINGGGSTYYPDSVKGRFTIS RDNARNILYLQMRSLRSEDTAMYYCARGGDYGYALDYWGQGTSVTVSS  >SEQ_ID_NO: 6 36A8 LC ENVLTQSPAIMSASLGEKVTMNCRASSSVNYMYWYQQKSDASPKLWIFYTSNLAPGVPARFSGSGSGNSY SLTISSMEGEDAATYYCQQFSSSPWTFGGGSKLEIKR  >SEQ_ID_NO: 7 4B11 LC EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLAWVASISSGGNIYFPDSVKGRFTIS RDDARNILYLQMRSLRSEDTAMYYCARGGDYGYAMDYWGQGTSVTVSS  >SEQ_ID_NO: 8 1H6 HC QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATL TADKSSSTAYMHLSSLTSDDSAVYFCARGRYEWYFDVWGAGTTVTVSS  >SEQ_ID_NO: 9 1H6 LC DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQRKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGYTLPYTFGGGTKLEIKR  >SEQ_ID_NO: 10 38A8 HC QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWIKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATL TADKSSSTAYMQLSSLTSDDSAVYFCARGRYEWYFDVWGAGTTVTVSS  >SEQ_ID_NO: 11 38A8 LC DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYLQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTD YSLSISNLEQEDIATYFCQQGYTLPYTFGGGTKLEIKR  >SEQ_ID_NO: 12 18F7 HC QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPPGKGLEWLGIIWGDGSTNYHSVLKSRLSIS KDNSKSQVFLKLNSLQTDDTATYYCAKQDFDVWGAGTTVTVSS  >SEQ_ID_NO: 13 18F7 LC DVQMIQSPFSLSASLGDIVTMTCQASQGTSINLNWFQQKPGKAPKLLIYGVSNLEDGVPSRFSGSRYGTD FTLTIGSLEDEDMATYFCLQHSYLPYTFGGGTKLEIKR  >SEQ_ID_NO: 14 12B2 HC QVQLQQSGAELTKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGITKYNDKFKGKATF TADTSSNTAYMQLSSLTSEDSAVYSCARLISYYYAMDYWGQGTSVTVSS  >SEQ_ID_NO: 15 12B2 LC DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIKR  >SEQ_ID_NO: 16 38F6 HC QVQLQQSGAELMRPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGTGYTKYNEKFKGKATF TAETSSNTASMQVSSLTSEDSAVYFCARLISYYYAMDYWGQGTSVTVSS  >SEQ_ID_NO: 17 5C4 HC QVTLKASGPGILQPSQTLSLTCSFSGFSLNTSGLGVGWIRQPSGKGLEWLAHIWWDDDKRYNPALKSRLT ISKDTSNNQIFLKIASVDTADTATYYCARSHYYGTFYFDYWGQGTTLTVSS  >SEQ_ID_NO: 18 23C10 HC FLLLIVPAYVLSQVTLKASGPGIVQPSQTLSLTCSFSGFSLNTSGMGVGWIRQPSGKGLEWLAHIWWDDD KRYNPALKSRLTISKDTSNNQIFLKIASVDTADTATYYCARSHYYGTFYFDYWGQGTTLTVSS  >SEQ_ID_NO: 19 28C2 HC QVTLKASGPGIVQPSQTLSLTCSFSGFSLNTSGMGVGWIRQPSGKGLEWLAHIWWDDDKRYNPALKSRLT ISKDTSNNQIFLKIASVDTADTATYYCARSHYYGTFYFDYWGQGTTLTVSS  >SEQ_ID_NO: 20 28C2 LC DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGTTYKLLIYSGSTLQSGIPSRFSGSGSGTD FTLTISSLEPEDFAMYYCQQHIEYPWTFGGGTKLEIKR  >SEQ_ID_NO: 21 9D6 HC QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQSSGKGLEWLAHIWWDDDKRYNPTLKSRLT ISKDTSNNQVFLKIANMDTADIATYYCARSHYNGTFYFDFWGQGITLTVSS  >SEQ_ID_NO: 22 9D6 LC DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTD FTLTISTLEPEDFAMYYCQQHIEYPWTFGGGTKLEIKR  >SEQ_ID_NO: 23 28F4 HC EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLEWVATISSGGTYTYYPDSVKGQFTI FRDNAKNTLYLQMSSLKSEDTAMYYCTRRDYDYEGFAYWGQGTLVTVS  >SEQ_ID_NO: 24 28F4 LC DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASHSISGIPSRFSGSGSGTD FTLSINSVETEDFGMYFCQQSNNWPFTFGSGTKLEIKR  >SEQ_ID_NO: 25 2A2 HCCDR1 TYAMS  >SEQ_ID_NO: 26 2A2 HCCDR2 SISSGSSTYYLDSVKG  >SEQ_ID_NO: 27 2A2 HCCDR3 GGDYGYALDY  >SEQ_ID_NO: 28 2A2 LCCDR1 RASSSVNYMY  >SEQ_ID_NO: 29 2A2 LCCDR2 YTSNLAP  >SEQ_ID_NO: 30 2A2 LCCDR3 QQFSSSPWT  >SEQ_ID_NO: 31 34D10 HCCDR1 AYAMS  >SEQ_ID_NO: 32 34D10 HCCDR2 SISSGGTTYYPDSVKR  >SEQ_ID_NO: 33 34D10 HCCDR3 GGDYGYALDY  >SEQ_ID_NO: 34 34D10 LCCDR1 RASSSVNYMY  >SEQ_ID_NO: 35 34D10 LCCDR2 YTSNLAP  >SEQ_ID_NO: 36 34D10 LCCDR3 QQFSSSPWT  >SEQ_ID_NO: 37 36A8 HCCDR1 TYAMS  >SEQ_ID_NO: 38 36A8 HCCDR2 SINGGGSTYYPDSVKG  >SEQ_ID_NO: 39 36A8 HCCDR3 GGDYGYALDY  >SEQ_ID_NO: 40 36A8 LCCDR1 RASSSVNYMY  >SEQ_ID_NO: 41 36A8 LCCDR2 YTSN LAP  >SEQ_ID_NO: 42 36A8 LCCDR3 QQFSSSPWT  >SEQ_ID_NO: 43 4B11 HCCDR1 SYAMS  >SEQ_ID_NO: 44 4B11 HCCDR2 SISSGGNIYFPDSVKG  >SEQ_ID_NO: 45 4B11 HCCDR3 GGDYGYAMDY  >SEQ_ID_NO: 46 1H6 HCCDR1  NYLIE  >SEQ_ID_NO: 47 1H6 HCCDR2 VINPGSGGTNYNEKFKG  >SEQ_ID_NO: 48 1H6 HCCDR3 GRYEWYFDV  >SEQ_ID_NO: 49 1H6 LCCDR1 RASQDITNYLN  >SEQ_ID_NO: 50 1H6 LCCDR2 YTSRLHS  >SEQ_ID_NO: 51 1H6 LCCDR3 QQGYTLPYT  >SEQ_ID_NO: 52 38A8 HCCDR1 NYLIE  >SEQ_ID_NO: 53 38A8 HCCDR2 VINPGSGGTNYNEKFKG  >SEQ_ID_NO: 54 38A8 HCCDR3 GRYEWYFDV  >SEQ_ID_NO: 55 38A8 LCCDR1 RASQDISNYLN  >SEQ_ID_NO: 56 38A8 LCCDR2 YTSRLHS  >SEQ_ID_NO: 57 38A8 LCCDR3 QQGYTLPYT  >SEQ_ID_NO: 58 18F7 HCCDR1 SYGVS  >SEQ_ID_NO: 59 18F7 HCCDR2 IIWGDGSTNYHSVLKS  >SEQ_ID_NO: 60 18F7 HCCDR3 QDFDV  >SEQ_ID_NO: 61 18F7 LCCDR1 QASQGTSINLN  >SEQ_ID_NO: 62 18F7 LCCDR2 GVSNLED  >SEQ_ID_NO: 63 18F7 LCCDR3 LQHSYLPYT  >SEQ_ID_NO: 64 12B2 HCCDR1 SYWIE  >SEQ_ID_NO: 65 12B2 HCCDR2 EILPGSGITKYNDKFKG  >SEQ_ID_NO: 66 12B2 HCCDR3 LISYYYAMDY  >SEQ_ID_NO: 67 12B2 LCCDR1 RASQDISNYLN  >SEQ_ID_NO: 68 12B2 LCCDR2 YTSRLHS  >SEQ_ID_NO: 69 12B2 LCCDR3 QQGNTLPPT  >SEQ_ID_NO: 70 38F6 HCCDR1 SYWIE  >SEQ_ID_NO: 71 38F6 HCCDR2 EILPGTGYTKYNEKFKG  >SEQ_ID_NO: 72 38F6 HCCDR3 LISYYYAMDY  >SEQ_ID_NO: 73 5C4 HCCDR1 TSGLGVG  >SEQ_ID_NO: 74 5C4 HCCDR2 HIWWDDDKRYNPALKS  >SEQ_ID_NO: 75 5C4 HCCDR3 SHYYGTFYFDY  >SEQ_ID_NO: 76 23C10 HCCDR1 TSGMGVG  >SEQ_ID_NO: 77 23C10 HCCDR2 HIWWDDDKRYNPALKS  >SEQ_ID_NO: 78 23C10 HCCDR3 SHYYGTFYFDY  >SEQ_ID_NO: 79 28C2 HCCDR1 TSGMGVG  >SEQ_ID_NO: 80 28C2 HCCDR2 HIWWDDDKRYNPALKS  >SEQ_ID_NO: 81 28C2 HCCDR3 SHYYGTFYFDY  >SEQ_ID_NO: 82 28C2 LCCDR1 RASKSISKYLA  >SEQ_ID_NO: 83 28C2 LCCDR2 SGSTLQS  >SEQ_ID_NO: 84 28C2 LCCDR3 QQHIEYPWT  >SEQ_ID_NO: 85 9D6 HCCDR1 TSGMGVG  >SEQ_ID_NO: 86 9D6 HCCDR2 HIWWDDDKRYNPTLKS  >SEQ_ID_NO: 87 9D6 HCCDR3 SHYNGTFYFDF  >SEQ_ID_NO: 88 9D6 LCCDR1 RASKSISKYLA  >SEQ_ID_NO: 89 9D6 LCCDR2 SGSTLQS  >SEQ_ID_NO: 90 9D6 LCCDR3 QQHIEYPWT  >SEQ_ID_NO: 91 28F4 HCCDR1 NYGMS  >SEQ_ID_NO: 92 28F4 HCCDR2 TISSGGTYTYYPDSVKG  >SEQ_ID_NO: 93 28F4 HCCDR3 RDYDYEGFAY  >SEQ_ID_NO: 94 28F4 LCCDR1 RASQSISNNLH  >SEQ_ID_NO: 95 28F4 LCCDR2 YASHSIS  >SEQ_ID_NO: 96 28F4 LCCDR3  QQSNNWPFT  >SEQ_ID_NO: 97 35D1 HC EVKLVESGGGLVKPGGSLKLSCAASGFTFSAYAMSWVRQTPEKRLEWVASISSGGTTYYPDSVKRRFTIS RDNARNILYLQMSSLRSEDTAMYYCTRGGDYGYALDYWGQGTSVTVSS  >SEQ_ID_NO: 98 35D1 LC ENVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSY SLTISSMEGEDAATYYCQQFSSSPWTFGGGTKLEIKR  >SEQ_ID_NO: 99 4B11 LC ENVLTQSPAIMSASLGEKVTMNCRASSSVNYMYWYQQKSDASPKLWIFYTSNLAPGVPARFSGSGSGNSY SLTISSMEGEDAATYYCQQFSSSPWTFGGGSKLEIKR  >SEQ_ID_NO: 100 38G8 HC QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATL TADKSSSTAYMHLSSLTSDDSAVYFCARGRYEWYFDVWGAGTTVTVSS  >SEQ_ID_NO: 101 38G8 LC DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQRKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGYTLPYTFGGGTKLEIKR  >SEQ_ID_NO: 102 21F10 HC DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQRKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGYTLPYTFGGGTKLEIKR  >SEQ_ID_NO: 103 21F10 LC DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQRKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGYTLPYTFGGGTKLEIKR  >SEQ_ID_NO: 104 38F6 LC* sequencing_pending  >SEQ_ID_NO: 105 13C1 HC QVQLQQSGAELTKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGITKYNDKFKGKATF TADTSSNTAYMQLSSLTSEDSAVYSCARLISYYYAMDYWGQGTSVTVSS  >SEQ_ID_NO: 106 13C1 LC sequencing_pending  >SEQ_ID_NO: 107 5C4 LC* sequencing_pending  >SEQ_ID_NO: 108 23C10 LC* sequencing_pending  >SEQ_ID_NO: 109 37C7 HC FLLLIVPAYVLSQVTLKASGPGIVQPSQTLSLTCSFSGFSLNTSGMGVGWIRQPSGKGLEWLAHIWWDDD KRYNPALKSRLTISKDTSNNQIFLKIASVDTADTATYYCARSHYYGTFYFDYWGQGTTLTVSS  >SEQ_ID_NO: 110 37C7 LC* sequencing_pending  >SEQ_ID_NO: 111 35D1 HCCDR1 AYAMS  >SEQ_ID_NO: 112 35D1 HCCDR2 SISSGGTTYYPDSVKR  >SEQ_ID_NO: 113 35D1 HCCDR3 GGDYGYALDY  >SEQ_ID_NO: 114 35D2 LCCDR1 RASSSVNYMY  >SEQ_ID_NO: 115 35D2 LCCDR2 YTSNLAP  >SEQ_ID_NO: 116 35D2 LCCDR3 QQFSSSPWT  >SEQ_ID_NO: 117 4B11 LCCDR1 RASSSVNYMY  >SEQ_ID_NO: 118 4B11 LCCDR2 YTSNLAP  >SEQ_ID_NO: 119 4B11 LCCDR3 QQFSSSPWT  >SEQ_ID_NO: 120 38G8 HCCDR1 NYLIE  >SEQ_ID_NO: 121 38G8 HCCDR2 VINPGSGGTNYNEKFKG  >SEQ_ID_NO: 122 38G8 HCCDR3 GRYEWYFDV  >SEQ_ID_NO: 123 38G8 LCCDR1 RASQDITNYLN  >SEQ_ID_NO: 124 38G8 LCCDR2 YTSRLHS  >SEQ_ID_NO: 125 38G8 LCCDR3 QQGYTLPYT  >SEQ_ID_NO: 126 21F10 HCCDR1 NYLIE  >SEQ_ID_NO: 127 21F10 HCCDR2 VINPGSGGTNYNEKFKG  >SEQ_ID_NO: 128 21F10 HCCDR3 GRYEWYFDV  >SEQ_ID_NO: 129 21F10 LCCDR1 RASQDITNYLN  >SEQ_ID_NO: 130 21F10 LCCDR2 YTSRLHS  >SEQ_ID_NO: 131 21F10 LCCDR3 QQGYTLPYT  >SEQ_ID_NO: 132 38F6 LC* CDR1 sequencing_pending  >SEQ_ID_NO: 133 38F6 LC* CDR2 sequencing_pending  >SEQ_ID_NO: 134 38F6 LC* CDR3 sequencing_pending  >SEQ_ID_NO: 135 13C1 HCCDR1 SYWIE  >SEQ_ID_NO: 136 13C1 HCCDR2 EILPGSGITKYNDKFKG  >SEQ_ID_NO: 137 13C1 HCCDR3 LISYYYAMDY  >SEQ_ID_NO: 138 13C1 LC* CDR1 sequencing_pending  >SEQ_ID_NO: 139 13C1 LC* CDR2 sequencing_pending  >SEQ_ID_NO: 140 13C1 LC* CDR3 sequencing_pending  >SEQ_ID_NO: 141 5C4 LC* CDR1 sequencing_pending  >SEQ_ID_NO: 142 5C4 LC* CDR2 sequencing_pending  >SEQ_ID_NO: 143 5C4 LC* CDR3 sequencing_pending  >SEQ_ID_NO: 144 23C10 LC* CDR1 sequencing_pending  >SEQ_ID_NO: 145 23C10 LC* CDR2 sequencing_pending  >SEQ_ID_NO: 146 23C10 LC* CDR3 sequencing_pending  >SEQ_ID_NO: 147 37C7 HCCDR1 TSGMGVG  >SEQ_ID_NO: 148 37C7 HCCDR2 HIWWDDDKRYNPALKS  >SEQ_ID_NO: 149 37C7 HCCDR3 SHYYGTFYFDY  >SEQ_ID_NO: 150 37C7 LC* CDR1 sequencing_pending  >SEQ_ID_NO: 151 37C7 LC* CDR2 sequencing_pending  >SEQ_ID_NO: 152 37C7 LC* CDR3  sequencing_pending SEQ ID NO: 153  >CTP peptide 1  DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL SEQ ID NO: 154  >CTP peptide 2  SSSSKAPPPSLPSPSRLPGPSDTPILPQ SEQ ID NO: 155  >PAS peptide 1  ASPAAPAPASPAAPAPSAPA SEQ ID NO: 156  >PAS peptide 2  AAPASPAPAAPSAPAPAAPS SEQ ID NO: 157  >PAS peptide 3  APSSPSPSAPSSPSPASPSS SEQ ID NO: 158  >PAS peptide 4  APSSPSPSAPSSPSPASPS SEQ ID NO: 159  >PAS peptide 5  SSPSAPSPSSPASPSPSSPA SEQ ID NO: 160  >PAS peptide 6  AASPAAPSAPPAAASPAAPSAPPA SEQ ID NO: 161  >PAS peptide 7  ASAAAPAAASAAASAPSAAA SEQ ID NO: 162  >Albumin Binding Peptide Core Sequence  DICLPRWGCLW SEQ ID NO: 163  >GFP protein sequence (Genbank ID AAG34521.1) MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKS AMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIE DGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSRTSGSPGLQEFDIKLIDTV DLESCN SEQ ID NO: 164  >Example: Single-chain Human IgG1 Fc. (Fc sequences with Gly/Ser linker underlined.) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 165  >Mature human albumin protein sequence (derived from NCBI Ref. Sequence NP 000468) RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFT ECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRH PDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLV EVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFH ADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL SEQ ID NO: 166  >Linker, n = 0, 1, 2, 3, 4 or more  (GGGS)_(n)SEQ ID NO: 167  >Albumin binding peptide 1  RLIEDICLPRWGCLWEDD SEQ ID NO: 168  >Albumin binding peptide 2  QRLMEDICLPRWGCLWEDDF SEQ ID NO: 169  >Albumin binding peptide 3  QGLIGDICLPRWGCLWGDSVK SEQ ID NO: 170  >Albumin binding peptide 4  GEWWEDICLPRWGCLWEEED SEQ ID NO: 171  >Cysteine-containing peptide  GGGSGCGGGS SEQ ID NO: 172  >Human LRP1 sequence (signal peptide and transmembrane segment underlined; NCBI Reference Sequence: CAA32112) MLTPPLLLLLPLLSALVAAAIDAPKTCSPKQFACRDQITCISKGWRCDGERDCPDGSDEAPEICPQSKAQRCQPNEHNCLGTELCV PMSRLCNGVQDCMDGSDEGPHCRELQGNCSRLGCQHHCVPTLDGPTCYCNSSFQLQADGKTCKDFDECSVYGTCSQLCTNTDGSFI CGCVEGYLLQPDNRSCKAKNEPVDRPPVLLIANSQNILATYLSGAQVSTITPTSTRQTTAMDFSYANETVCWVHVGDSAAQTQLKC ARMPGLKGFVDEHTINISLSLHHVEQMAIDWLTGNFYFVDDIDDRIFVCNRNGDTCVTLLDLELYNPKGIALDPAMGKVFFTDYGQ IPKVERCDMDGQNRTKLVDSKIVFPHGITLDLVSRLVYWADAYLDYIEVVDYEGKGRQTIIQGILIEHLYGLTVFENYLYATNSDN ANAQQKTSVIRVNRFNSTEYQVVTRVDKGGALHIYHQRRQPRVRSHACENDQYGKPGGCSDICLLANSHKARTCRCRSGFSLGSDG KSCKKPEHELFLVYGKGRPGIIRGMDMGAKVPDEHMIPIENLMNPRALDFHAETGFIYFADTTSYLIGRQKIDGTERETILKDGIH NVEGVAVDWMGDNLYWTDDGPKKTISVARLEKAAQTRKTLIEGKMTHPRAIVVDPLNGWMYWTDWEEDPKDSRRGRLERAWMDGSH RDIFVTSKTVLWPNGLSLDIPAGRLYWVDAFYDRIETILLNGTDRKIVYEGPELNHAFGLCHHGNYLFWTEYRSGSVYRLERGVGG APPTVTLLRSERPPIFEIRMYDAQQQQVGTNKCRVNNGGCSSLCLATPGSRQCACAEDQVLDADGVTCLANPSYVPPPQCQPGEFA CANSRCIQERWKCDGDNDCLDNSDEAPALCHQHTCPSDRFKCENNRCIPNRWLCDGDNDCGNSEDESNATCSARTCPPNQFSCASG RCIPISWTCDLDDDCGDRSDESASCAYPTCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSSTQFKCNSGRCIPEHW TCDGDNDCGDYSDETHANCTNQATRPPGGCHTDEFQCRLDGLCIPLRWRCDGDTDCMDSSDEKSCEGVTHVCDPSVKFGCKDSARC ISKAWVCDGDNDCEDNSDEENCESLACRPPSHPCANNTSVCLPPDKLCDGNDDCGDGSDEGELCDQCSLNNGGCSHNCSVAPGEGI VCSCPLGMELGPDNHTCQIQSYCAKHLKCSQKCDQNKFSVKCSCYEGWVLEPDGESCRSLDPFKPFIIFSNRHEIRRIDLHKGDYS VLVPGLRNTIALDFHLSQSALYWTDVVEDKIYRGKLLDNGALTSFEVVIQYGLATPEGLAVDWIAGNIYWVESNLDQIEVAKLDGT LRTTLLAGDIEHPRAIALDPRDGILFWTDWDASLPRIEAASMSGAGRRTVHRETGSGGWPNGLTVDYLEKRILWIDARSDAIYSAR YDGSGHMEVLRGHEFLSHPFAVTLYGGEVYWTDWRTNTLAKANKWTGHNVTVVQRTNTQPFDLQVYHPSRQPMAPNPCEANGGQGP CSHLCLINYNRTVSCACPHLMKLHKDNTTCYEFKKFLLYARQMEIRGVDLDAPYYNYIISFTVPDIDNVTVLDYDAREQRVYWSDV RTQAIKRAFINGTGVETVVSADLPNAHGLAVDWVSRNLFWTSYDTNKKQINVARLDGSFKNAVVQGLEQPHGLVVHPLRGKLYWTD GDNISMANMDGSNRTLLFSGQKGPVGLAIDFPESKLYWISSGNHTINRCNLDGSGLEVIDAMRSQLGKATALAIMGDKLWWADQVS EKMGTCSKADGSGSVVLRNSTTLVMHMKVYDESIQLDHKGTNPCSVNNGDCSQLCLPTSETTRSCMCTAGYSLRSGQQACEGVGSF LLYSVHEGIRGIPLDPNDKSDALVPVSGTSLAVGIDFHAENDTIYWVDMGLSTISRAKRDQTWREDVVTNGIGRVEGIAVDWIAGN IYWTDQGFDVIEVARLNGSFRYVVISQGLDKPRAITVHPEKGYLFWTEWGQYPRIERSRLDGTERVVLVNVSISWPNGISVDYQDG KLYWCDARTDKIERIDLETGENREVVLSSNNMDMFSVSVFEDFIYWSDRTHANGSIKRGSKDNATDSVPLRTGIGVQLKDIKVFNR DRQKGTNVCAVANGGCQQLCLYRGRGQRACACAHGMLAEDGASCREYAGYLLYSERTILKSIHLSDERNLNAPVQPFEDPEHMKNV IALAFDYRAGTSPGTPNRIFFSDIHFGNIQQINDDGSRRITIVENVGSVEGLAYHRGWDTLYWTSYTTSTITRHTVDQTRPGAFER ETVITMSGDDHPRAFVLDECQNLMFWTNWNEQHPSIMRAALSGANVLTLIEKDIRTPNGLAIDHRAEKLYFSDATLDKIERCEYDG SHRYVILKSEPVHPFGLAVYGEHIFWTDWVRRAVQRANKHVGSNMKLLRVDIPQQPMGIIAMANDTNSCELSPCRINNGGCQDLCL LTHQGHVNCSCRGGRILQDDLTCRAVNSSCRAQDEFECANGECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNGRCVS NMLWCNGADDCGDGSDEIPCNKTACGVGEFRCRDGTCIGNSSRCNQFVDCEDASDEMNCSATDCSSYFRLGVKGVLFQPCERTSLC YAPSWVCDGANDCGDYSDERDCPGVKRPRCPLNYFACPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISKQWL CDGSDDCGDGSDEAAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGADESIAAGCLYNSTCDDREFMCQNRQCIPKHFVCD HDRDCADGSDESPECEYPTCGPSEFRCANGRCLSSRQWECDGENDCHDQSDEAPKNPHCTSPEHKCNASSQFLCSSGRCVAEALLC NGQDDCGDSSDERGCHINECLSRKLSGCSQDCEDLKIGFKCRCRPGFRLKDDGRTCADVDECSTTFPCSQRCINTHGSYKCLCVEG YAPRGGDPHSCKAVTDEEPFLIFANRYYLRKLNLDGSNYTLLKQGLNNAVALDFDYREQMIYWTDVTTQGSMIRRMHLNGSNVQVL HRTGLSNPDGLAVDWVGGNLYWCDKGRDTIEVSKLNGAYRTVLVSSGLREPRALVVDVQNGYLYWTDWGDHSLIGRIGMDGSSRSV IVDTKITWPNGLTLDYVTERIYWADAREDYIEFASLDGSNRHVVLSQDIPHIFALTLFEDYVYWTDWETKSINRAHKTTGTNKTLL ISTLHRPMDLHVFHALRQPDVPNHPCKVNNGGCSNLCLLSPGGGHKCACPTNFYLGSDGRTCVSNCTASQFVCKNDKCIPFWWKCD TEDDCGDHSDEPPDCPEFKCRPGQFQCSTGICTNPAFICDGDNDCQDNSDEANCDIHVCLPSQFKCTNTNRCIPGIFRCNGQDNCG DGEDERDCPEVTCAPNQFQCSITKRCIPRVWVCDRDNDCVDGSDEPANCTQMTCGVDEFRCKDSGRCIPARWKCDGEDDCGDGSDE PKEECDERTCEPYQFRCKNNRCVPGRWQCDYDNDCGDNSDEESCTPRPCSESEFSCANGRCIAGRWKCDGDHDCADGSDEKDCTPR CDMDQFQCKSGHCIPLRWRCDADADCMDGSDEEACGTGVRTCPLDEFQCNNTLCKPLAWKCDGEDDCGDNSDENPEECARFVCPPN RPFRCKNDRVCLWIGRQCDGTDNCGDGTDEEDCEPPTAHTTHCKDKKEFLCRNQRCLSSSLRCNMFDDCGDGSDEEDCSIDPKLTS CATNASICGDEARCVRTEKAAYCACRSGFHTVPGQPGCQDINECLRFGTCSQLCNNTKGGHLCSCARNFMKTHNTCKAEGSEYQVL YIADDNEIRSLFPGHPHSAYEQAFQGDESVRIDAMDVHVKAGRVYWTNWHTGTISYRSLPPAAPPTTSNRHRRQIDRGVTHLNISG LKMPRGIAIDWVAGNVYWTDSGRDVIEVAQMKGENRKTLISGMIDEPHAIVVDPLRGTMYWSDWGNHPKIETAAMDGTLRETLVQD NIQWPTGLAVDYHNERLYWADAKLSVIGSIRLNGTDPIVAADSKRGLSHPFSIDVFEDYIYGVTYINNRVFKIHKFGHSPLVNLTG GLSHASDVVLYHQHKQPEVTNPCDRKKCEWLCLLSPSGPVCTCPNGKRLDNGTCVPVPSPTPPPDAPRPGTCNLQCFNGGSCFLNA RRQPKCRCQPRYTGDKCELDQCWEHCRNGGTCAASPSGMPTCRCPTGFTGPKCTQQVCAGYCANNSTCTVNQGNQPQCRCLPGFLG DRCQYRQCSGYCENFGTCQMAADGSRQCRCTAYFEGSRCEVNKCSRCLEGACVVNKQSGDVTCNCTDGRVAPSCLTCVGHCSNGGS CTMNSKMMPECQCPPHMTGPRCEEHVFSQQQPGHIASILIPLLLLLLLVLVAGVVFWYKRRVQGAKGFQHQRMTNGAMNVEIGNPT YKMYEGGEPDDVGGLLDADFALDPDKPTNFTNPVYATLYMGGHGSRHSLASTDEKRELLGRGPEDEIGDPLA SEQ ID NO: 173  >Biotin Acceptor Peptide (BAP)  LNDIFEAQKIEWH SEQ ID NO: 174  >Lipoate Acceptor Peptide 2 (LAP2)  GFEIDKVWYDLDA SEQ ID NO: 175  >HAPylation motif, n = 1 to 400  (Gly4Ser)n SEQ ID NO: 176  >Alternative linker  PEAPTDPEAPTD  SEQ ID NO: 177  >CTP DSSSSKAPPPSLPSPSRLPGPSDTPILPQ  SEQ ID NO: 178  >FVII-HC IVGGKVCP KGECPWQVLL LVNGAQLCGG TLINTIWVVS AAHCFDKIKN WRNLIAVLGE HDLSEHDGDE QSRRVAQVII PSTYVPGTTN HDIALLRLHQ PVVLTDHVVP LCLPERTFSE RTLAFVRFSL VSGWGQLLDR GATALELMVL NVPRLMTQDC LQQSRKVGDS PNITEYMFCA GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL MRSEPRPGVL LRAPFP SEQ ID NO: 179  >FVII-LC ANAFLEELRP GSLERECKEE QCSFEEAREI FKDAERTKLF WISYSDGDQC ASSPCQNGGS CKDQLQSYIC FCLPAFEGRN CETHKDDQLI CVNENGGCEQ YCSDHTGTKR SCRCHEGYSL LADGVSCTPT VEYPCGKIPI LEKRNASKPQ GR  SEQ ID NO: 180  >FVII zymogen. MVSQALRLLC LLLGLQGCLA AGGVAKASGG ETRDMPWKPG PHRVFVTQEE AHGVLHRRRR ANAFLEELRP GSLERECKEE QCSFEEAREI FKDAERTKLF WISYSDGDQC ASSPCQNGGS CKDQLQSYIC FCLPAFEGRN CETHKDDQLI CVNENGGCEQ YCSDHTGTKR SCRCHEGYSL LADGVSCTPT VEYPCGKIPI LEKRNASKPQ GRIVGGKVCP KGECPWQVLL LVNGAQLCGG TLINTIWVVS AAHCFDKIKN WRNLIAVLGE HDLSEHDGDE QSRRVAQVII PSTYVPGTTN HDIALLRLHQ PVVLTDHVVP LCLPERTFSE RTLAFVRFSL VSGWGQLLDR GATALELMVL NVPRLMTQDC LQQSRKVGDS PNITEYMFCA GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL MRSEPRPGVL LRAPFP SEQ ID NO: 181  >FIX zymogen. Signal sequence (1-28). Propeptide (29-46) MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTE FWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEP AVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVN EKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNI FLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT SEQ ID NO: 182  >FX zymogen. Signal sequence (1-23). Propeptide (24-40) MGRPLHLVLLSASLAGLLLLGESLFIRREQANNILARVTRANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKD GDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCELFTRKLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADNGKACIPTGPYPC GKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTRIVGGQECKDGECPWQALLINEENE GFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLP ERDWAESTLMTQKTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGPHVTRFKDT YFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSSPLK SEQ ID NO: 183  >Human GPIIb. Signal sequence (1-31). Transmembrane (981-1019). Cytoplasmic (1020- 1039) MARALCPLQALWLLEWVLLLLGPCAAPPAWALNLDPVQLTFYAGPNGSQFGFSLDFHKDSHGRVAIVVGAPRTLGPSQEETGGVFL CPWRAEGGQCPSLLFDLRDETRNVGSQTLQTFKARQGLGASVVSWSDVIVACAPWQHWNVLEKTEEAEKTPVGSCFLAQPESGRRA EYSPCRGNTLSRIYVENDFSWDKRYCEAGFSSVVTQAGELVLGAPGGYYFLGLLAQAPVADIFSSYRPGILLWHVSSQSLSFDSSN PEYFDGYWGYSVAVGEFDGDLNTTEYVVGAPTWSWTLGAVEILDSYYQRLHRLRGEQMASYFGHSVAVTDVNGDGRHDLLVGAPLY MESRADRKLAEVGRVYLFLQPRGPHALGAPSLLLTGTQLYGRFGSAIAPLGDLDRDGYNDIAVAAPYGGPSGRGQVLVFLGQSEGL RSRPSQVLDSPFPTGSAFGFSLRGAVDIDDNGYPDLIVGAYGANQVAVYRAQPVVKASVQLLVQDSLNPAVKSCVLPQTKTPVSCF NIQMCVGATGHNIPQKLSLNAELQLDRQKPRQGRRVLLLGSQQAGTTLNLDLGGKHSPICHTTMAFLRDEADFRDKLSPIVLSLNV SLPPTEAGMAPAVVLHGDTHVQEQTRIVLDCGEDDVCVPQLQLTASVTGSPLLVGADNVLELQMDAANEGEGAYEAELAVHLPQGA HYMRALSNVEGFERLICNQKKENETRVVLCELGNPMKKNAQIGIAMLVSVGNLEEAGESVSFQLQIRSKNSQNPNSKIVLLDVPVR AEAQVELRGNSFPASLVVAAEEGEREQNSLDSWGPKVEHTYELHNNGPGTVNGLHLSIHLPGQSQPSDLLYILDIQPQGGLQCFPQ PPVNPLKVDWGLPIPSPSPIHPAHHKRDRRQIFLPEPEQPSRLQDPVLVSCDSAPCTVVQCDLQEMARGQRAMVTVLAFLWLPSLY QRPLDQFVLQSHAWFNVSSLPYAVPPLSLPRGEAQVWTQLLRALEERAIPIWWVLVGVLGGLLLLTILVLAMWKVGFFKRNRPPLE EDDEEGE SEQ ID NO: 184  >Human GPIIIa. Signal sequence (1-26) . Transmembrane (719-747) . Cytoplasmic (748-788) MRARPRPRPLWATVLALGALAGVGVGGPNICTTRGVSSCQQCLAVSPMCAWCSDEALPLGSPRCDLKENLLKDNCAPESIEFPVSE ARVLEDRPLSDKGSGDSSQVTQVSPQRIALRLRPDDSKNFSIQVRQVEDYPVDIYYLMDLSYSMKDDLWSIQNLGTKLATQMRKLT SNLRIGFGAFVDKPVSPYMYISPPEALENPCYDMKTTCLPMFGYKHVLTLTDQVTRFNEEVKKQSVSRNRDAPEGGFDAIMQATVC DEKIGWRNDASHLLVFTTDAKTHIALDGRLAGIVQPNDGQCHVGSDNHYSASTTMDYPSLGLMTEKLSQKNINLIFAVTENVVNLY QNYSELIPGTTVGVLSMDSSNVLQLIVDAYGKIRSKVELEVRDLPEELSLSFNATCLNNEVIPGLKSCMGLKIGDTVSFSIEAKVR GCPQEKEKSFTIKPVGFKDSLIVQVTFDCDCACQAQAEPNSHRCNNGNGTFECGVCRCGPGWLGSQCECSEEDYRPSQQDECSPRE GQPVCSQRGECLCGQCVCHSSDFGKITGKYCECDDFSCVRYKGEMCSGHGQCSCGDCLCDSDWTGYYCNCTTRTDTCMSSNGLLCS GRGKCECGSCVCIQPGSYGDTCEKCPTCPDACTFKKECVECKKFDRGALHDENTCNRYCRDEIESVKELKDTGKDAVNCTYKNEDD CVVRFQYYEDSSGKSILYVVEEPECPKGPDILVVLLSVMGAILLIGLAALLIWKLLITIHDRKEFAKFEEERARAKWDTANNPLYK EATSTFTNITYRGT  SEQ ID NO: 185  >Human GPIIb DNA. ATGGCCAGAGCTTTGTGTCCACTGCAAGCCCTCTGGCTTCTGGAGTGGGTGCTGCTGCTCTACCGGTCTCGAAACACAGGTGACGT TCGGGAGACCGAAGACCTCACCCACGACGACGAGTTGGGACCTTGTGCTGCCCCTCCAGCCTGGGCCTTGAACCTGGACCCAGTGC AGCTCACCAACCCTGGAACACGACGGGGAGGTCGGACCCGGAACTTGGACCTGGGTCACGTCGAGTGGTTCTATGCAGGCCCCAAT GGCAGCCAGTTTGGATTTTCACTGGACTTCCACAAGGACAGCAAGATACGTCCGGGGTTACCGTCGGTCAAACCTAAAAGTGACCT GAAGGTGTTCCTGTCGCATGGGAGAGTGGCCATCGTGGTGGGCGCCCCGCGGACCCTGGGCCCCAGCCAGGAGGAGGTACCCTCTC ACCGGTAGCACCACCCGCGGGGCGCCTGGGACCCGGGGTCGGTCCTCCTCACGGGCGGCGTGTTCCTGTGCCCCTGGAGGGCCGAG GGCGGCCAGTGCCCCTCGCTGCTCTGCCCGCCGCACAAGGACACGGGGACCTCCCGGCTCCCGCCGGTCACGGGGAGCGACGAGTT TGACCTCCGTGATGAGACCCGAAATGTAGGCTCCCAAACTTTACAAACCTTCAAGGCCAAACTGGAGGCACTACTCTGGGCTTTAC ATCCGAGGGTTTGAAATGTTTGGAAGTTCCGGCGCCAAGGACTGGGGGCGTCGGTCGTCAGCTGGAGCGACGTCATTGTGGCCTGC GCCCCCGCGGTTCCTGACCCCCGCAGCCAGCAGTCGACCTCGCTGCAGTAACACCGGACGCGGGGGTGGCAGCACTGGAACGTCCT AGAAAAGACTGAGGAGGCTGAGAAGACGCCCGTAGGTAGCACCGTCGTGACCTTGCAGGATCTTTTCTGACTCCTCCGACTCTTCT GCGGGCATCCATCGTGCTTTTTGGCTCAGCCAGAGAGCGGCCGGCGCGCCGAGTACTCCCCCTGTCGCGGGAACACGAAAAACCGA GTCGGTCTCTCGCCGGCCGCGCGGCTCATGAGGGGGACAGCGCCCTTGACCCTGAGCCGCATTTACGTGGAAAATGATTTTAGCTG GGACAAGCGTTACTGTGAAGCGTGGGACTCGGCGTAAATGCACCTTTTACTAAAATCGACCCTGTTCGCAATGACACTTCGCGGCT TCAGCTCCGTGGTCACTCAGGCCGGAGAGCTGGTGCTTGGGGCTCCTGGCGGCTATCCGAAGTCGAGGCACCAGTGAGTCCGGCCT CTCGACCACGAACCCCGAGGACCGCCGATATATTTCTTAGGTCTCCTGGCCCAGGCTCCAGTTGCGGATATTTTCTCGAGTTACCG CCCAATAAAGAATCCAGAGGACCGGGTCCGAGGTCAACGCCTATAAAAGAGCTCAATGGCGGGTGGCATCCTTTTGTGGCACGTGT CCTCCCAGAGCCTCTCCTTTGACTCCAGCAACCCAGAGCCGTAGGAAAACACCGTGCACAGGAGGGTCTCGGAGAGGAAACTGAGG TCGTTGGGTCTCTACTTCGACGGCTACTGGGGGTACTCGGTGGCCGTGGGCGAGTTCGACGGGGATCTCAACATGAAGCTGCCGAT GACCCCCATGAGCCACCGGCACCCGCTCAAGCTGCCCCTAGAGTTGACTACAGAATATGTCGTCGGTGCCCCCACTTGGAGCTGGA CCCTGGGAGCGGTGGAAATTTGATGTCTTATACAGCAGCCACGGGGGTGAACCTCGACCTGGGACCCTCGCCACCTTTAATTGGAT TCCTACTACCAGAGGCTGCATCGGCTGCGCGGAGAGCAGATGGCGTCGTATTTTAACCTAAGGATGATGGTCTCCGACGTAGCCGA CGCGCCTCTCGTCTACCGCAGCATAAAAGGGCATTCAGTGGCTGTCACTGACGTCAACGGGGATGGGAGGCATGATCTGCTGGTGG GCCCCGTAAGTCACCGACAGTGACTGCAGTTGCCCCTACCCTCCGTACTAGACGACCACCCGGCTCCACTGTATATGGAGAGCCGG GCAGACCGAAAACTGGCCGAAGTGGGGCGTGTGTATCGAGGTGACATATACCTCTCGGCCCGTCTGGCTTTTGACCGGCTTCACCC CGCACACATATTGTTCCTGCAGCCGCGAGGCCCCCACGCGCTGGGTGCCCCCAGCCTCCTGCTGACTGGCAACAAGGACGTCGGCG CTCCGGGGGTGCGCGACCCACGGGGGTCGGAGGACGACTGACCGACACAGCTCTATGGGCGATTCGGCTCTGCCATCGCACCCCTG GGCGACCTCGACCGGGATTGTGTCGAGATACCCGCTAAGCCGAGACGGTAGCGTGGGGACCCGCTGGAGCTGGCCCTAGGCTACAA TGACATTGCAGTGGCTGCCCCCTACGGGGGTCCCAGTGGCCGGGGCCAAGTGCCGATGTTACTGTAACGTCACCGACGGGGGATGC CCCCAGGGTCACCGGCCCCGGTTCACCTGGTGTTCCTGGGTCAGAGTGAGGGGCTGAGGTCACGTCCCTCCCAGGTCCTGGACAGC GACCACAAGGACCCAGTCTCACTCCCCGACTCCAGTGCAGGGAGGGTCCAGGACCTGTCGCCCTTCCCCACAGGCTCTGCCTTTGG CTTCTCCCTTCGAGGTGCCGTAGACATCGATGACGGGAAGGGGTGTCCGAGACGGAAACCGAAGAGGGAAGCTCCACGGCATCTGT AGCTACTGAACGGATACCCAGACCTGATCGTGGGAGCTTACGGGGCCAACCAGGTGGCTGTGTACAGATTGCCTATGGGTCTGGAC TAGCACCCTCGAATGCCCCGGTTGGTCCACCGACACATGTCTGCTCAGCCAGTGGTGAAGGCCTCTGCCAGCTACTGGTGCAAGAT TCACTGAATCCTGCTCGAGTCGGTCACCACTTCCGGAGACAGGTCGATGACCACGTTCTAAGTGACTTAGGACGAGTGAAGAGCTG TGTCCTACCTCAGACCAAGACACCCGTGAGCTGCTTCAACATCCAGATGCACTTCTCGACACAGGATGGAGTCTGGTTCTGTGGGC ACTCGACGAAGTTGTAGGTCTACTGTGTTGGAGCCACTGGGCACAACATTCCTCAGAAGCTATCCCTAAATGCCGAGCTGCAGACA CAACCTCGGTGACCCGTGTTGTAAGGAGTCTTCGATAGGGATTTACGGCTCGACGTCCTGGACCGGCAGAAGCCCCGCCAGGGCCG GCGGGTGCTGCTGCTGGGCTCTCAACAGGCAGACCTGGCCGTCTTCGGGGCGGTCCCGGCCGCCCACGACGACGACCCGAGAGTTG TCCGTGGCACCACCCTGAACCTGGATCTGGGCGGAAAGCACAGCCCCATCTGCCACACCACCATGCCGTGGTGGGACTTGGACCTA GACCCGCCTTTCGTGTCGGGGTAGACGGTGTGGTGGTACGCCTTCCTTCGAGATGAGGCAGACTTCCGGGACAAGCTGAGCCCCAT TGTGCTCAGCCTCCGGAAGGAAGCTCTACTCCGTCTGAAGGCCCTGTTCGACTCGGGGTAACACGAGTCGGAGAATGTGTCCCTAC CGCCCACGGAGGCTGGAATGGCCCCTGCTGTCGTGCTGCATGGAGACTTACACAGGGATGGCGGGTGCCTCCGACCTTACCGGGGA CGACAGCACGACGTACCTCTGACCCATGTGCAGGAGCAGACACGAATCGTCCTGGACTGTGGGGAAGATGACGTATGTGTGTGGGT ACACGTCCTCGTCTGTGCTTAGCAGGACCTGACACCCCTTCTACTGCATACACACCCCCAGCTTCAGCTCACTGCCAGCGTGACGG GCTCCCCGCTCCTAGTTGGGGCAGATAATGGGGTCGAAGTCGAGTGACGGTCGCACTGCCCGAGGGGCGAGGATCAACCCCGTCTA TTAGTCCTGGAGCTGCAGATGGACGCAGCCAACGAGGGCGAGGGGGCCTATGAAGCAGAGCTGCAGGACCTCGACGTCTACCTGCG TCGGTTGCTCCCGCTCCCCCGGATACTTCGTCTCGACGCCGTGCACCTGCCCCAGGGCGCCCACTACATGCGGGCCCTAAGCAATG TCGAGGGCTTTCGGCACGTGGACGGGGTCCCGCGGGTGATGTACGCCCGGGATTCGTTACAGCTCCCGAAAGAGAGACTCATCTGT AATCAGAAGAAGGAGAATGAGACCAGGGTGGTGCTGTGTGAGCTGCTCTCTGAGTAGACATTAGTCTTCTTCCTCTTACTCTGGTC CCACCACGACACACTCGACGGCAACCCCATGAAGAAGAACGCCCAGATAGGAATCGCGATGTTGGTGAGCGTGGGGAATCCGTTGG GGTACTTCTTCTTGCGGGTCTATCCTTAGCGCTACAACCACTCGCACCCCTTACTGGAAGAGGCTGGGGAGTCTGTGTCCTTCCAG CTGCAGATACGGAGCAAGAACAGCCAGGACCTTCTCCGACCCCTCAGACACAGGAAGGTCGACGTCTATGCCTCGTTCTTGTCGGT CAATCCAAACAGCAAGATTGTGCTGCTGGACGTGCCGGTCCGGGCAGAGGCCCAAGTGGAGTTAGGTTTGTCGTTCTAACACGACG ACCTGCACGGCCAGGCCCGTCTCCGGGTTCACCTCCTGCGAGGGAACTCCTTTCCAGCCTCCCTGGTGGTGGCAGCAGAAGAAGGT GAGAGGGAGGACGCTCCCTTGAGGAAAGGTCGGAGGGACCACCACCGTCGTCTTCTTCCACTCTCCCTCCAGAACAGCTTGGACAG CTGGGGACCCAAAGTGGAGCACACCTATGAGCTCCACAACAATGTCTTGTCGAACCTGTCGACCCCTGGGTTTCACCTCGTGTGGA TACTCGAGGTGTTGTTAGGCCCTGGGACTGTGAATGGTCTTCACCTCAGCATCCACCTTCCGGGACAGTCCCAGCCCCCGGGACCC TGACACTTACCAGAAGTGGAGTCGTAGGTGGAAGGCCCTGTCAGGGTCGGGTCCGACCTGCTCTACATCCTGGATATACAGCCCCA GGGGGGCCTTCAGTGCTTCCCACAGAGGCTGGACGAGATGTAGGACCTATATGTCGGGGTCCCCCCGGAAGTCACGAAGGGTGTCC CTCCTGTCAACCCTCTCAAGGTGGACTGGGGGCTGCCCATCCCCAGCCCCTCCCCCATTGGAGGACAGTTGGGAGAGTTCCACCTG ACCCCCGACGGGTAGGGGTCGGGGAGGGGGTAACACCCGGCCCATCACAAGCGGGATCGCAGACAGATCTTCCTGCCAGAGCCCGA GCAGCCCGTGGGCCGGGTAGTGTTCGCCCTAGCGTCTGTCTAGAAGGACGGTCTCGGGCTCGTCGGGTCGAGGCTTCAGGATCCAG TTCTCGTAAGCTGCGACTCGGCGCCCTGTACTGTGGTGCAGAGCTCCGAAGTCCTAGGTCAAGAGCATTCGACGCTGAGCCGCGGG ACATGACACCACGTCTGTGACCTGCAGGAGATGGCGCGCGGGCAGCGGGCCATGGTCACGGTGCTGGCCTTCCTGACACTGGACGT CCTCTACCGCGCGCCCGTCGCCCGGTACCAGTGCCACGACCGGAAGGACTGGCTGCCCAGCCTCTACCAGAGGCCTCTGGATCAGT TTGTGCTGCAGTCGCACGCATGGACCGACGGGTCGGAGATGGTCTCCGGAGACCTAGTCAAACACGACGTCAGCGTGCGTACCTTC AACGTGTCCTCCCTCCCCTATGCGGTGCCCCCGCTCAGCCTGCCCCGAGGGGAAGCTAAGTTGCACAGGAGGGAGGGGATACGCCA CGGGGGCGAGTCGGACGGGCTCCCCTTCGACAGGTGTGGACACAGCTGCTCCGGGCCTTGGAGGAGAGGGCCATTCCAATCTGGTG GGTGGTCCACACCTGTGTCGACGAGGCCCGGAACCTCCTCTCCCGGTAAGGTTAGACCACCCACCTGGTGGGTGTGCTGGGTGGCC TGCTGCTGCTCACCATCCTGGTCCTGGCCATGTGGAAGGACCACCCACACGACCCACCGGACGACGACGAGTGGTAGGACCAGGAC CGGTACACCTTCGTCGGCTTCTTCAAGCGGAACCGGCCACCCCTGGAAGAAGATGATGAAGAGGGGGAGTGACAGCCGAAGAAGTT CGCCTTGGCCGGTGGGGACCTTCTTCTACTACTTCTCCCCCTCACT SEQ ID NO: 186  >Human GPIIIa DNA ATGCGAGCGCGCCCGCGGCCCCGGCCGCTCTGGGCGACTGTGCTGGCGCTGGGGGCGCTGTACGCTCGCGCGGGCGCCGGGGCCGG CGAGACCCGCTGACACGACCGCGACCCCCGCGACGCGGGCGTTGGCGTAGGAGGGCCCAACATCTGTACCACGCGAGGTGTGAGCT CCTGCCAGCGCCCGCAACCGCATCCTCCCGGGTTGTAGACATGGTGCGCTCCACACTCGAGGACGGTCCAGTGCCTGGCTGTGAGC CCCATGTGTGCCTGGTGCTCTGATGAGGCCCTGCCTCTGGGCGTCACGGACCGACACTCGGGGTACACACGGACCACGAGACTACT CCGGGACGGAGACCCGTCACCTCGCTGTGACCTGAAGGAGAATCTGCTGAAGGATAACTGTGCCCCAGAATCCATCAGTGGAGCGA CACTGGACTTCCTCTTAGACGACTTCCTATTGACACGGGGTCTTAGGTAGGAGTTCCCAGTGAGTGAGGCCCGAGTACTAGAGGAC AGGCCCCTCAGCGACAAGGGCTCTCTCAAGGGTCACTCACTCCGGGCTCATGATCTCCTGTCCGGGGAGTCGCTGTTCCCGAGAGG AGACAGCTCCCAGGTCACTCAAGTCAGTCCCCAGAGGATTGCACTCCGGCTCCGGCCACCTCTGTCGAGGGTCCAGTGAGTTCAGT CAGGGGTCTCCTAACGTGAGGCCGAGGCCGGTGATGATTCGAAGAATTTCTCCATCCAAGTGCGGCAGGTGGAGGATTACCCTGTG GACATCCTACTAAGCTTCTTAAAGAGGTAGGTTCACGCCGTCCACCTCCTAATGGGACACCTGTAGTACTACTTGATGGACCTGTC TTACTCCATGAAGGATGATCTGTGGAGCATCCAGAACCTGATGATGAACTACCTGGACAGAATGAGGTACTTCCTACTAGACACCT CGTAGGTCTTGGACGGTACCAAGCTGGCCACCCAGATGCGAAAGCTCACCAGTAACCTGCGGATTGGCTTCGGGCCATGGTTCGAC CGGTGGGTCTACGCTTTCGAGTGGTCATTGGACGCCTAACCGAAGCCCGCATTTGTGGACAAGCCTGTGTCACCATACATGTATAT CTCCCCACCAGAGGCCCTCGAACGTAAACACCTGTTCGGACACAGTGGTATGTACATATAGAGGGGTGGTCTCCGGGAGCTTAACC CCTGCTATGATATGAAGACCACCTGCTTGCCCATGTTTGGCTACAAACACGTGCTGTTGGGGACGATACTATACTTCTGGTGGACG AACGGGTACAAACCGATGTTTGTGCACGACACGCTAACTGACCAGGTGACCCGCTTCAATGAGGAAGTGAAGAAGCAGAGTGTGTC ACGGTGCGATTGACTGGTCCACTGGGCGAAGTTACTCCTTCACTTCTTCGTCTCACACAGTGCCAACCGAGATGCCCCAGAGGGTG GCTTTGATGCCATCATGCAGGCTACAGTCTGTGATGAATTGGCTCTACGGGGTCTCCCACCGAAACTACGGTAGTACGTCCGATGT CAGACACTACTTAAGATTGGCTGGAGGAATGATGCATCCCACTTGCTGGTGTTTACCACTGATGCCAAGACTTTCTAACCGACCTC CTTACTACGTAGGGTGAACGACCACAAATGGTGACTACGGTTCTGACATATAGCATTGGACGGAAGGCTGGCAGGCATTGTCCAGC CTAATGACGGGCAGTGTCATGTATATCGTAACCTGCCTTCCGACCGTCCGTAACAGGTCGGATTACTGCCCGTCACAGTAGTTGGT AGTGACAATCATTACTCTGCCTCCACTACCATGGATTATCCCTCTTTGGGGCTGCAACCATCACTGTTAGTAATGAGACGGAGGTG ATGGTACCTAATAGGGAGAAACCCCGACATGACTGAGAAGCTATCCCAGAAAAACATCAATTTGATCTTTGCAGTGACTGAAAATG TATACTGACTCTTCGATAGGGTCTTTTTGTAGTTAAACTAGAAACGTCACTGACTTTTACATGTCAATCTCTATCAGAACTATAGT GAGCTCATCCCAGGGACCACAGTTGGGGTTCTGTCCCAGTTAGAGATAGTCTTGATATCACTCGAGTAGGGTCCCTGGTGTCAACC CCAAGACAGGATGGATTCCAGCAATGTCCTCCAGCTCATTGTTGATGCTTATGGGAAAATCCGTTCTAAATACCTAAGGTCGTTAC AGGAGGTCGAGTAACAACTACGAATACCCTTTTAGGCAAGATTTGTAGAGCTGGAAGTGCGTGACCTCCCTGAAGAGTTGTCTCTA TCCTTCAATGCCACCTGCCATCTCGACCTTCACGCACTGGAGGGACTTCTCAACAGAGATAGGAAGTTACGGTGGACGCTCAACAA TGAGGTCATCCCTGGCCTCAAGTCTTGTATGGGACTCAAGATTGGAGACACGGAGTTGTTACTCCAGTAGGGACCGGAGTTCAGAA CATACCCTGAGTTCTAACCTCTGTGCGTGAGCTTCAGCATTGAGGCCAAGGTGCGAGGCTGTCCCCAGGAGAAGGAGAAGTCCTTT CACTCGAAGTCGTAACTCCGGTTCCACGCTCCGACAGGGGTCCTCTTCCTCTTCAGGAAAACCATAAAGCCCGTGGGCTTCAAGGA CAGCCTGATCGTCCAGGTCACCTTTGATTGTGACTGGTATTTCGGGCACCCGAAGTTCCTGTCGGACTAGCAGGTCCAGTGGAAAC TAACACTGTGTGCCTGCCAGGCCCAAGCTGAACCTAATAGCCATCGCTGCAACAATGGCAATGGGACCACACGGACGGTCCGGGTT CGACTTGGATTATCGGTAGCGACGTTGTTACCGTTACCCTGGTTTGAGTGTGGGGTATGCCGTTGTGGGCCTGGCTGGCTGGGATC CCAGTGTGAGTGCTCAAAACTCACACCCCATACGGCAACACCCGGACCGACCGACCCTAGGGTCACACTCACGAGTGAGGAGGACT ATCGCCCTTCCCAGCAGGACGAATGCAGCCCCCGGGAGGGTCAGCCCGTCCTCCTCCTGATAGCGGGAAGGGTCGTCCTGCTTACG TCGGGGGCCCTCCCAGTCGGGCAGTGCAGCCAGCGGGGCGAGTGCCTCTGTGGTCAATGTGTCTGCCACAGCAGTGACTTTGGCAC GTCGGTCGCCCCGCTCACGGAGACACCAGTTACACAGACGGTGTCGTCACTGAAACCGAAGATCACGGGCAAGTACTGCGAGTGTG ACGACTTCTCCTGTGTCCGCTACAAGGGGGAGTTCTAGTGCCCGTTCATGACGCTCACACTGCTGAAGAGGACACAGGCGATGTTC CCCCTCATGTGCTCAGGCCATGGCCAGTGCAGCTGTGGGGACTGCCTGTGTGACTCCGACTGGACCTACACGAGTCCGGTACCGGT CACGTCGACACCCCTGACGGACACACTGAGGCTGACCTGGGGCTACTACTGCAACTGTACCACGCGTACTGACACCTGCATGTCCA GCAATGGGCTGCTGCCGATGATGACGTTGACATGGTGCGCATGACTGTGGACGTACAGGTCGTTACCCGACGACTGCAGCGGCCGG GGCAAGTGTGAATGTGGCAGCTGTGTCTGTATCCAGCCGGGCTCCTATACGTCGCCGGCCCCGTTCACACTTACACCGTCGACACA GACATAGGTCGGCCCGAGGATAGGGGACACCTGTGAGAAGTGCCCCACCTGCCCAGATGCCTGCACCTTTAAGAAAGAATGTCCCC TGTGGACACTCTTCACGGGGTGGACGGGTCTACGGACGTGGAAATTCTTTCTTACAGTGGAGTGTAAGAAGTTTGACCGGGGAGCC CTACATGACGAAAATACCTGCAACCGTTACCACCTCACATTCTTCAAACTGGCCCCTCGGGATGTACTGCTTTTATGGACGTTGGC AATGTGCCGTGACGAGATTGAGTCAGTGAAAGAGCTTAAGGACACTGGCAAGGATGCAGTGAATACGGCACTGCTCTAACTCAGTC ACTTTCTCGAATTCCTGTGACCGTTCCTACGTCACTTATGTACCTATAAGAATGAGGATGACTGTGTCGTCAGATTCCAGTACTAT GAAGATTCTAGTACATGGATATTCTTACTCCTACTGACACAGCAGTCTAAGGTCATGATACTTCTAAGATCAGGAAAGTCCATCCT GTATGTGGTAGAAGAGCCAGAGTGTCCCAAGGGCCCTGACATCCTGCCTTTCAGGTAGGACATACACCATCTTCTCGGTCTCACAG GGTTCCCGGGACTGTAGGACGTGGTCCTGCTCTCAGTGATGGGGGCCATTCTGCTCATTGGCCTTGCCGCCCTGCTCATCCACCAG GACGAGAGTCACTACCCCCGGTAAGACGAGTAACCGGAACGGCGGGACGAGTAGTGGAAACTCCTCATCACCATCCACGACCGAAA AGAGTTCGCTAAATTTGAGGAAGAACGCACCTTTGAGGAGTAGTGGTAGGTGCTGGCTTTTCTCAAGCGATTTAAACTCCTTCTTG CGGCCAGAGCAAAATGGGACACAGCCAACAACCCACTGTATAAAGAGGCCACGTCTACCTTCCGGTCTCGTTTTACCCTGTGTCGG TTGTTGGGTGACATATTTCTCCGGTGCAGATGGAAGACCAATATCACGTACCGGGGCACTTAATGGTTATAGTGCATGGCCCCGTG AATT 

1. An antibody or antigen-binding molecule thereof which specificallybinds to a GPIIb/IIIa epitope, (a) wherein the antibody orantigen-binding molecule thereof specifically bind to the sameGPIIb/IIIa epitope as an antibody selected from 34D10, 12B2, 2A2, 35D1,36A8, 4B11, 1H6, 38G8, 21F10, 38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7,28C2, 9D6, or 28F4; (b) wherein the antibody or antigen-binding moleculethereof competitively inhibits GPIIb/IIIa binding by an antibodyselected from 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10,38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4; or (c)wherein the antibody or antigen-binding molecule thereof comprises atleast one, at least two, at least three, at least four, or at least fivecomplementarity determining regions (CDR) or variants thereof of anantibody selected from the 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6,38G8, 21F10, 38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or28F4 antibodies.
 2. The antibody or antigen-binding molecule thereof ofclaim 1, which comprises six CDRs or variants thereof of an antibodyselected from the 34D10, 12B2, 2A2, 35D1, 36A8, 4B11, 1H6, 38G8, 21F10,38A8, 18F7, 38F6, 13C1, 5C4, 23C10, 37C7, 28C2, 9D6, or 28F4 antibodies.3. (canceled)
 4. An antibody or antigen-binding molecule thereof whichspecifically binds to a GPIIb/IIIa epitope, comprising (i) a VH-CDR1comprising the consensus sequence XIYAMS wherein X₁ represents aminoacid residues Thr (T), Ser (S), or Ala (A); (ii) a VH-CDR2 comprisingthe consensus sequence SIX₂X₃GX₄X₅TYX₆X₇DSVKX₈ wherein X₂ representsamino acid residues Ser (S) or Asn (N), X₃ represents amino acidresidues Ser (S) or Gly (G), X₄ represents amino acid residues Ser (S)or Gly (G), X₅ represents amino acid residues Ser (S), Asn (N), or Thr(T), X₆ represents amino acid residues Tyr (Y) or Phe (F), X₇ representsamino acid residues Leu (L) or Pro (P), and X₈ represents amino acidsGly (G) or Arg (R); (iii) a VH-CDR3 comprising the consensus sequenceGGDYGYAX₉DY, wherein X₉ represents amino acid residues Leu (L) or Met(M); (iv) a VL-CDR1 comprising the sequence RASSSVNYMY (SEQ ID NO: 28);(v) a VL-CDR2 comprising the sequence YTSNLAP (SEQ ID NO: 29); and, (vi)a VL-CDR3 comprising the sequence QQFSSSPWT (SEQ ID NO: 30).
 5. Theantibody or antigen-binding molecule thereof according to claim 4,comprising (i) a VH-CDR1 sequence selected from SEQ ID NOS: 25, 31, 37,43, or 111; (ii) a VH-CDR2 sequence selected from SEQ ID NOS: 26, 32,38, 44, or 112; (iii) a VH-CDR3 sequence selected from SEQ ID NOS: 27,33, 39, 45, or 113; (iv) a VL-CDR1 sequence selected from SEQ ID NOS:28, 34, 40, 117, or 114; (v) a VL-CDR2 sequence selected from f SEQ IDNOS: 29, 35, 41, 118, or 115; and, (vi) a VL-CDR3 sequence selected fromSEQ ID NOS: 30, 36, 42, 119, or
 116. 6-53. (canceled)
 54. The antibodyor antigen-binding molecule thereof of claim 1, wherein the antibody orantigen-binding molecule thereof comprises or consists of: (a) a singlechain Fv (“scFv”); (b) a diabody; (c) a minibody; (d) a polypeptidechain of an antibody; (e) F(ab′)₂; or, (f) F(ab).
 55. A chimericmolecule comprising, (i) the antibody or antigen-binding moleculethereof of claim 1; (ii) a heterologous moiety; and, (iii) an optionallinker.
 56. (canceled)
 57. The chimeric molecule according to claim 55,wherein the heterologous moiety comprises a clotting factor.
 58. Thechimeric molecule according to claim 57, wherein the clotting factor isindependently selected from the group consisting of FVII, FIX, FX, andany combinations thereof.
 59. The chimeric molecule according to claim58, wherein the clotting factor is FVII zymogen, activatable FVII,activated FVII (FVIIa), FIX zymogen, activatable FIX, activated FIX(FIXa), FX zymogen, activatable FX, or activated FX (FXa).
 60. Thechimeric molecule according to claim 55, further comprising a secondheterologous moiety.
 61. The chimeric molecule according to claim 60,which comprises a formula selected from: (i) H1-(L1)-Tm-(L2)-H2; (ii)H2-(L2)-Tm-(L1)-H1; (iii) H1-(L1)-H2-(L2)-Tm; (iv) H2-(L2)-H1-(L1)-Tm;(v) Tm-(L1)-H1-(L2)-H2; or, (vi) Tm-(L2)-H2-(L1)-H1 wherein, Tm is theantibody or antigen-binding molecule thereof of claim 1; H1 is the firstheterologous moiety, H2 is the second heterologous moiety, L1 is thefirst optional linker, L2 is the second optional linker.
 62. (canceled)63. The chimeric molecule according to claim 60, wherein the secondheterologous moiety comprise a half-life extending moiety. 64.-68.(canceled)
 69. A chimeric protein comprising a first polypeptide chainand a second polypeptide chain, which are associated with each other,(1) wherein the first polypeptide chain comprises a light chain of aclotting factor and a heterologous moiety, and the second polypeptidechain comprises a heavy chain of the clotting factor and the antibody orantigen-binding molecule thereof of claim 1; (2) wherein the firstpolypeptide chain comprises a light chain of a clotting factor and theantibody or antigen-binding molecule thereof of claim 1 and the secondpolypeptide chain comprises a heavy chain of the clotting factor and aheterologous moiety; (3) wherein the first polypeptide chain comprises alight chain of a clotting factor and the second polypeptide chaincomprises a heavy chain of the clotting factor, the antibody orantigen-binding molecule thereof of claim 1, and a heterologous moiety;(4) wherein the first polypeptide chain comprises a light chain of aclotting factor and the second polypeptide chain comprises a heavy chainof the clotting factor, a heterologous moiety, and the antibody orantigen-binding molecule thereof of claim 1; (5) wherein the firstpolypeptide chain comprises a light chain of a clotting factor, aheterologous moiety, and the antibody or antigen-binding moleculethereof of claim 1 and the second polypeptide chain comprises a heavychain of the clotting factor; or (6) wherein the first polypeptide chaincomprises a light chain of a clotting factor, the antibody orantigen-binding molecule thereof of claim 1, and a heterologous moietyand the second polypeptide chain comprises a heavy chain of the clottingfactor. 70-76. (canceled)
 77. The chimeric molecule according to claim69, wherein the heterologous moiety comprise a half-life extendingmoiety comprising a low-complexity polypeptide. 78-81. (canceled)
 82. Achimeric molecule comprising a first polypeptide chain and a secondpolypeptide chain, which are associated with each other, (1) wherein thefirst polypeptide chain comprises a light chain of a clotting factor anda targeting moiety, which binds to a platelet, and the secondpolypeptide chain comprises a heavy chain of the clotting factor and aheterologous moiety; (2) wherein the first polypeptide chain comprises alight chain of a clotting factor and a heterologous moiety and thesecond polypeptide chain comprises a heavy chain of the clotting factorand a targeting moiety, which binds to a platelet; (3) wherein the firstpolypeptide chain comprises a light chain of a clotting factor, aheterologous moiety, and a targeting moiety, which binds to a platelet,and the second polypeptide comprises a heavy chain of the clottingfactor; or (4) wherein the first polypeptide chain comprises a lightchain of a clotting factor, a targeting moiety, which binds to aplatelet, and a heterologous moiety and the second polypeptide chaincomprises a heavy chain of the clotting factor, and wherein the clottingfactor is FVII, FIX, or FX.
 83. The chimeric molecule of claim 82, (1)wherein the first polypeptide chain comprises CF_(L)-L1-H or H-L1-CF_(L)and the second polypeptide chain comprises CF_(H)-L2-Tm or Tm-L2-CF_(H);(2) wherein the first polypeptide chain comprises CF_(L)-L1-Tm orTm-L1-CF_(L) and the second polypeptide chain comprises CF_(H)-L2-H orH-L2-CF_(H); (3) wherein the first polypeptide chain comprisesCF_(L)-L1-H-L2-Tm or Tm-L1-H-L2-CF_(L) and the second polypeptide chaincomprises CF_(H); or (4) wherein the first polypeptide chain comprisesCF_(L)-L1-Tm-L2-H or H-L1-Tm-L2-CF_(L) and the second polypeptide chaincomprises CF_(H); wherein H is the heterologous moiety; CF_(H) is theheavy chain of the clotting factor; CF_(L) is the light chain of theclotting factor; Tm is the targeting moiety, which binds to a platelet;L1 is a first optional linker; and L2 is a second optional linker.84-99. (canceled)
 100. A nucleic acid molecule or a set of nucleic acidmolecules encoding an antibody or antigen-binding molecule thereof ofclaim 1 or a complement thereof.
 101. A vector or a set of vectorscomprising the nucleic acid molecule or the set of the nucleic acidmolecules of claim 100 or a complement thereof.
 102. A host cellcomprising the vector according to claim
 101. 103. A method forproducing an antibody or antigen-binding molecule thereof comprisingculturing the host cell according to claim 102 and recovering theantibody or antigen-binding molecule thereof from the culture medium.104. A pharmaceutical composition comprising the antibody orantigen-binding molecule thereof of claim 1 and a pharmaceuticallyacceptable carrier.
 105. A method of reducing a frequency or degree of ableeding episode in a subject in need thereof comprising administeringthe antibody or antigen-binding molecule thereof of claim
 1. 106-108.(canceled)
 109. The method according to claim 105, wherein the bleedingepisode is caused by a blood coagulation disorder. 110-114. (canceled)115. A method to target a therapeutic or prophylactic agent to thesurface of platelets comprising fusing the agent to the antibody orantigen-binding molecule thereof according to claim
 1. 116. A method toincrease the activity of a therapeutic or prophylactic agent comprisingfusing the agent to the antibody or antigen-binding molecule thereofaccording to claim
 1. 117. (canceled)
 118. A method to improve thepharmacokinetic properties of a clotting factor comprising fusing theclotting factor to the antibody or antigen-binding molecule thereofaccording to claim
 1. 119. (canceled)
 120. A method of measuring thelevel of platelets in plasma of a subject in need thereof comprisingcontacting the antibody or antigen binding molecule thereof of claim 1with the plasma from the subject and measuring the plasma level.